Alcohols, C16-18 and C18-unsatd., 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) = 300 mg/kg bw/day

 

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

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

 

Conclusion based on data obtained with alcohols, C16-18 and C18 unsatd., ethoxylated (CAS No. 68920-66-1, EC No. 500-016-2) 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:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to the category justification provided in the category object.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

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

For a detailed assessment of the repeated dose toxicity of the Alcohol Ethoxylates (AE) category, please refer to the category justification attached to the category object.

Conclusions:

Applying read-across based on grouping of substances (category approach), no toxicologically relevant effects after short-term repeated dose administration and a NOAEL for systemic toxicity ≥ 1000 mg/kg bw/day are predicted for the registered substance.

Executive summary:

The available data on repeated dose toxicity in the 'linear' subgroup of the Alcohol Ethoxylates (AE) category indicates no toxicologically relevant effects for the registered substance. As explained in the category justification, the differences in molecular structure and composition between the registered substance and the members of the AE category are unlikely to lead to differences with respect to short-term repeated dose toxicity.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Jul 2021 - 12 Jan 2022

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 and humidity outside targeted range for 3 days (T = 17 °C, H = 39%) - blood urea nitrogen not evaluated during clinical biochemistry analysis

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar (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: 140 - 202 g (males) and 104 - 148 g (females)
- Fasting period before study: no
- Housing: Up to 5 animals of the same sex and dosing group were 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, Germany) and equipped with water bottles. Animals were provided with enrichment items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. During locomotor activity monitoring, animals were 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 rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); ad libitum (except during motor activity measurements, where food is withheld for a maximum of 2 h)
- Water: municipal tap water provided via water bottles; ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
Routine analysis of the diet and water showed no indication for known contaminants that could interfere with the outcomes of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 39 - 68
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From 09 Jul - 17 Nov 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations (w/w) were homogenized to visually acceptable levels, prepared at weekly intervals and stored at 4°C. No adjustments for specific gravity, nor for purity/composition were made. The dose formulations were stirred continuously during dosing and administered using plastic feeding tubes. The dose volume was 4 mL/kg bw/day, adjusted to the animal's most recent body weight measurement.

VEHICLE
- Justification for use and choice of vehicle: trial preparations were performed outside the scope of the present study to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 25 mg/mL (100 mg/kg bw/day), 75 mg/mL (300 mg/kg bw/day), 250 mg/mL (1000 mg/kg bw/day)
- Amount of vehicle: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis at Weeks 1, 6 and 12. For concentration analysis, 2x 500 mg samples were taken from the middle of the dosing container for all groups. For homogeneity analysis, 2x 500 mg samples were collected from top, middle and bottom of the dosing container for Groups 2 and 4, each. Analyses for stability, concentration and homogeneity were performed by using a validated analytical procedure.
Method: Gas chromatographic method with flame ionization detection (GC-FID)
Results: For all occasions, the concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90 - 110% of target concentration; acceptance criteria ± 15% of theoretical concentration). No test item was detected in the Group 1 formulations. For all occasions, the formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤10%).

Duration of treatment / exposure:

90 days + 28 days recovery period (control and high-dose recovery groups)

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:

10 (main study)
5 (recovery control and high-dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 14-day repeated dose toxicity study and in an attempt to produce graded responses to the test item.
- 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: checks for mortality were performed at least twice daily beginning upon arrival through termination, except on days of receipt and necropsy where frequency was at least once daily. Cage side observations were performed at least once daily from Day 1 at 0 - 1 h post-dose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration, and weekly during the treatment period

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)

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 (only qualitatively checked)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the pretreatment period, and once during Week 13
- Dose groups that were examined: all animals during pretreatment period, and all group 1 and 4 animals during Week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment or recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight with a maximum of 24 h
- How many animals: all main study and recovery animals
- Parameters checked: white blood cells, absolute counts of neutrophils, lymphocytes, monocytes, eosinophils, basophils, reticulocytes and large unstained cells, red blood cells, red blood cell distribution width, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, prothrombin time, and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment or recovery period
- Animals fasted: Yes, overnight with a maximum of 24 h
- How many animals: all main study and recovery animals
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, triglycerides, HDL and LDL cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, triiodothyronine, thyroxine, and thyroid-stimulating hormone.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at the end of treatment or recovery period
- Animals fasted: Yes, overnight with a maximum of 24 h
- How many animals: all main study and recovery animals

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment or recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight with a maximum of 24 h
- Parameters checked: volume and pH

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

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment or recovery period, all animals surviving until scheduled euthanasia had their terminal body weight recorded and were then deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy. Animals were subjected to a complete necropsy examination, including 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.

ORGAN WEIGHTS: Yes
The following organs were weighed from all surviving animals of the treatment or recovery groups: brain, epididymis, adrenal gland, pituitary gland, prostate, seminal vesicles, thyroid, heart, kidney, liver, ovary, spleen, testis, thymus, and uterus/cervix.

HISTOPATHOLOGY: Yes
Representative samples of tissues were collected and preserved in 10% neutral buffered formalin or modified Davidson's solution. Tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. For treatment groups 1 and 4, the following list of organs/tissues was microscopically assessed:
artery (aorta), bone marrow (sternum), brain, bone (sternum), epididymis, esophagus, eye, adrenal gland, mammary gland, parathyroid gland, pituitary gland, prostate, salivary gland (submandibular and sublingual), seminal vesicle (including coagulation gland), thyroid, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph node (mandibular and mesenteric), skeletal muscle, optic nerve, sciatic nerve, ovary, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testis, thymus, trachea, urinary bladder, uterus/cervix, and vagina.
For treatment groups 2 and 3, as well as recovery groups 1 and 4, only gross lesions and target tissues were evaluated.

Other examinations:

ESTROUS STAGE DETERMINATION
At the end of treatment (day of necropsy), a vaginal smear was taken to determine the stage of estrus from all females. This was done for all females, except for females that have to be euthanized in extremis or died spontaneously. The estrous stage was evaluated by examining the vaginal cytology of the samples obtained by vaginal smears procedures.

SPERM ANALYSIS
For all surviving males, sperm samples were taken from the proximal part of the right vas deferens at necropsy. Sperm count and sperm motility, and progressive motility were evaluated from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa per animal were recorded. Additionally, the right epididymis of each animal was removed, weighed, homogenized and evaluated for sperm numbers. In case of abnormalities noted, the organ was fixed in modified Davidson's solution and the left epididymis used for sperm evaluation instead.

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were 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 applied: Levene's test, one-way ANOVA F-test, Kruskal-Wallis test, Dunnett's test, Dunn's test, ANCOVA, and Fisher's exact test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the dosing period, incidental abnormal breathing sounds were observed in 1/10 males at 100 mg/kg bw/day, 1/10 males and 1/10 females at 300 mg/kg bw/day and in 6/10 males and 4/10 females at 1000 mg/kg bw/day. Salivation after dosing was observed at all dose levels (except for females at 100 mg/kg bw/day) at a dose-related incidence. The effects were considered to be a physiological response to the gavage treatment and not toxicologically relevant.
During the recovery period, only abnormal breathing sounds were noted once (Day 108) in 1/5 males at 1000 mg/kg bw/day.
Any other clinical signs 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 or occurred in the control group. 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 - Table 1 under "Overall remarks, attachments".

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality occurred in two females at 1000 mg/kg bw/day.
Both females were found dead on Day 51 before dosing. Abnormal breathing sounds were observed in one of the females on Days 36 - 40 and Day 43. No clinical signs were observed in the other female and no effect on body weight was observed in either of the females. For both animals, microscopic findings of neutrophilic inflammation in the surface of the heart (epicardial) and lungs (pleura) and in one female, focal transmural inflammation of the esophagus, was consistent with a gavage accident as the cause of death, and therefore, not a cause of the test substance itself. In the other female, however, there was also accumulation of pale wispy eosinophilic material (presumed test item) in the larger airways, suggestive of gastric reflux and aspiration. Similar observations were noted in scheduled euthanized animals. Examination of the caudal nasal cavities and larynx was performed for the second female found dead and only minimal exudates were observed in the larynx and ventral nasal cavity. The mortality of that female was suspected to be treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was considered to be unaffected by treatment with the test item in males at 100 mg/kg bw/day and in females at all dose levels.
In males at 300 mg/kg bw/day, a slightly lower body weight was observed from Day 43 onwards. Males treated at 1000 mg/kg bw/day showed lower body weight from Day 22 onwards. Throughout the recovery period, body weights in males at 1000 mg/kg bw/day remained decreased (-22%) at end of recovery period.
In males at 300 mg/kg bw/day, a lower body weight gain compared to controls between Days 36 and 43 was noticed, which resulted in a 6.8% lower body weight compared to the controls at the end of the dosing period (not statistically significant). Males treated at 1000 mg/kg bw/day showed an overall lower body weight gain throughout the dosing period as well. This resulted in a 15.5% lower body weight compared to controls at the end of the dosing period. Throughout the recovery period, body weights in males at 1000 mg/kg bw/day remained decreased (-22% at end of recovery period). The body weight effects at 1000 mg/kg bw/day were of a higher magnitude and did not recover during the recovery period and were therefore considered to be adverse.
A decreased and increased body weight gain compared to the controls was observed in females between Days 22 - 29 at 300 mg/kg bw/day and 43 - 50 at 1000 mg/kg bw/day, respectively. At the incidence observed it was considered not test item related.
For details on body weight and body weight gain, please refer to Attachments 1 - Tables 2 - 3 and Attachment 2 - Figure 1 under "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was considered to be unaffected by treatment with the test item in males at 100 mg/kg bw/day and in females at all dose levels.
Males treated at 300 and 1000 mg/kg bw/day showed a slightly lower food consumption throughout the dosing period, resulting in a 5.5% and 6.9% lower food consumption during the complete dosing period (Day 1 - 91), respectively, compared to controls. Food consumption in males at 1000 mg/kg bw/day remained lower throughout the recovery period (-21% at end of recovery period).
For details on food consumption, please refer to Attachments 1 - Table 4 and Attachment 2 - Figure 2 under "Overall remarks, attachments".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The nature and incidence of ophthalmology findings noted during the pretreatment period and in Week 13 was similar among the groups and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to the test item.
For details on ophthalmological examinations, please refer to Attachment 3 under "Overall remarks, attachments".

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters were considered unaffected by treatment with the test item in males up to 1000 mg/kg bw/day and in females up to 300 mg/kg bw/day.
Females treated at 1000 mg/kg bw/day showed increased white blood cell (1.32 x, p≤0.05), neutrophil (2.56 x, p≤0.01), and monocyte (1.99 x, p≤0.01) counts at the end of the dosing period, compared to controls (see Table 1 under section "Any other information on results incl. tables"). These changes were no longer present at the end of the recovery period. In absence of a microscopic correlate and at the magnitude of change observed, these reversible findings were considered not adverse.
Remaining differences in hematology parameters, 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, slightly low or high control values, were of a magnitude of change commonly observed in rats under similar study conditions and/or were solely seen at end of recovery.
Coagulation parameters were considered unaffected by treatment with the test item in males and females. The shorter prothrombin time in males treated at 300 mg/kg bw/day was without a dose-related response and was therefore considered to be unrelated to treatment with the test item.
For details on haematology and coagulation, please refer to Attachment 1 - Tables 7 - 8 under "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry parameters were considered unaffected by treatment with the test item in males and in females up to 100 mg/kg bw/day.
At 1000 mg/kg bw/day, alanine aminotransferase (1.72 x (males) and 1.36 x (females), p≤0.01) and aspartate aminotransferase activity (1.31 x (males), p≤0.01), and cholesterol (1.33 x (males) and 1.35 x (females), p≤0.01), HDL cholesterol (1.13 x (males) and 1.25 x (females), p≤0.05) and LDL cholesterol (1.60 x (males) and 1.48 x (females), p≤0.01 in females only) concentrations were increased in males and/or females at 1000 mg/kg bw/day, compared to controls. In addition, at 300 mg/kg bw/day increased urea concentrations (1.22 x (females), p≤0.05) and calcium concentrations (1.07 x (males), p≤0.05) were observed at end of the dosing period, compared to controls. Calcium levels remained increased (1.07 x, not statistically significant) in males at 1000 mg/kg bw/day. These changes were no longer present at end of the recovery period. For details on affected clinical chemistry changes, please refer to Table 2 under section "Any other information on results incl. tables."
Remaining differences in clinical chemistry parameters 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, slightly low or high control values, were of a magnitude of change commonly observed in rats under similar study conditions and/or were solely seen at end of recovery.
For details on clinical biochemistry, please refer to Attachment 1 - Table 9 under "Overall remarks, attachments".

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Thyroid stimulating hormone (TSH) concentrations were increased in males at 1000 mg/kg bw/day (1.52 x of controls), and in females at all dose levels (2.59 x, 1.53 x and 1.95 x, of controls, respectively). At end of the recovery period, TSH concentrations showed an opposite effect compared to end of the dosing period in males, whereas in females, TSH concentrations were increased compared to the end of the dosing period (4.26 x of controls). These findings were likely to be test item related, though not considered adverse as the values fell within the historical control data range. All group means remained within the range of historical control data (Historical Control Data for Wistar Han rats (2015-2021): TSH (mU/L) – males: mean = 0.1591; P5-P95: 0.0260-0.7253 (n=208); TSH (mU/L) – females: mean = 0.0871; P5-P95: 0.0020-0.3994 (n=204))
For details on Hormone Analysis, please refer to Attachment 1 - Table 9 under "Overall remarks, attachments".

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine parameters were unaffected by treatment with the test item in males and females.
For details on urinalysis, please refer to Attachment 1 - Table 10 under "Overall remarks, attachments."

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between test item-treated and control groups. 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 functional observations, please refer to Attachment 1 - Tables 5 - 6 under "Overall remarks, attachments".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related organ weight differences were noted at 1000 mg/kg bw/day at the end of the dosing period in the liver of males and females and in the adrenal gland of males.
Males: At the end of the dosing period, relatively large differences in terminal body weight were noted (-13%) at 1000 mg/kg bw/day. At the same dose, higher liver weights were observed relative to body weight only (+14%). While there may be an effect of the lower body weight on this apparent difference, the evaluation of the relative (to body) weight of this organ is the preferred parameter to evaluate the effects of a test chemical on the liver weight (Bailey et al., 2004) and this small difference may represent a test item-related effect. At the end of the recovery period, terminal body weights at 1000 mg/kg bw/day were still reduced (-23%), but relative to body weight liver weights were not affected anymore (recovered), however, it is noteworthy that the body weight and absolute liver weight were both -23% compared to the controls.
Females: At the end of the dosing period, higher liver weights were observed at 1000 mg/kg bw/day (absolute value +16%; relative to body weight value: +15%; the latter being statistically significant) without an appreciable difference in terminal body weights or macroscopic/microscopic correlates. No effect on liver weights was noted at the end of the recovery period, indicating recovery of the effects occurring during the dosing period.
To sum up, the higher liver weights noted in males and females at 1000 mg/kg bw/day were of small magnitude, without histologic correlate, and recovered, therefore were considered non-adverse.
In the adrenal gland, at the end of the dosing period in males at 1000 mg/kg bw/day, higher weight was noted as absolute and relative values. There was no macroscopic or microscopic correlate. Differences noted at 300 mg/kg bw/day were considered not to be test item-related because of the low magnitude change as an absolute value and statistical significance relative to body weight only. Since the adrenal gland exhibits a non-proportional relationship to body weight, this is not the preferred parameter for evaluation of this organ (Bailey et al., 2004). The higher adrenal gland weight in males at 1000 mg/kg bw/day may be secondary to chronic stress, and was considered non-adverse.
No adrenal gland weight difference was observed at the end of the recovery period (recovered). No adrenal gland weight difference was observed in females at the end of the treatment or recovery period. For details on liver and adrenal gland weights, please refer to Table 3 under section "Any other information on results incl. tables."
There were no other organ weight differences considered to be test item-related.
At the end of the dosing period in males at 1000 mg/kg bw/day several organs which are generally considered to remain stable in the face of body weight fluctuation were apparently higher in weight when expressed relative to body weight only. This was considered to be secondary to the low terminal body weight (-13%) and included the brain (+15%), heart (+8%), testes (+14%), and epididymis (+13%). Similarly, in the recovery males at 1000 mg/kg bw/day several organs were apparently higher in weight when expressed relative to body weight only and were also considered to be secondary to the low body weight (-23%) including the brain (+22%), pituitary gland (+12%), kidney (+12%), spleen (+30%), testes (+26%), and epididymis (+35%).
Lower prostate weights were noted in males at 300 and 1000 mg/kg bw/day at the end of the dosing period but were significant only as absolute values and lacked a dose related trend and were therefore not considered test item-related. No prostate weight difference was noted at the end of the recovery period. Lower absolute heart weight of recovery males was statistically significant (-17%) but interpreted as likely secondary to the lower body weight/smaller size of the animals in this group (-23%).
Higher uterus weights were noted in females at 1000 mg/kg bw/day at the end of the recovery period compared to the control recovery females and were interpreted to be due to differences in estrous stage and were not considered test item-related.
For details on organ weights, please refer to Attachment 1 - Tables 12 -13 under "Overall remarks, attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related dark foci was observed in the glandular mucosa of the stomach in the 1000 mg/kg bw/day group males (4/10) and correlated to minimal foci of mucosal hemorrhage. The single incidence at 300 mg/kg bw/day was considered to be within the range of background.
The few remaining recorded macroscopic findings at the end of the dosing and recovery period were within the range of background gross observations encountered in rats of this age and strain.
For details on macroscopic observations, please refer to Attachment 1- Table 14 under "Overall remarks, attachments".

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 stomach, jejunum and/or duodenum, trachea and/or lung of males and females, kidney of males and hematopoietic tissues (bone marrow and spleen) of females:
In the non-glandular stomach, hyperplasia of the squamous mucosa was noted in 7/10 males (up to mild) and 6/9 females (minimal) at 1000 mg/kg bw/day. This was also noted at minimal degree in a single female at 300 mg/kg bw/day but at this low incidence and severity is not strong evidence of a test item-related effect.
In males, a low incidence of acute mucosal/submucosal hemorrhage of the glandular portion of the stomach (minimal, 3/10 males) was also noted at 1000 mg/kg bw/day and correlated with dark foci noted macroscopically. This was also noted at minimal degree in a single male at 300 mg/kg bw/day but at this low incidence and severity is not strong evidence of a test item-related effect.
Following the recovery period, minimal hyperplasia of the squamous mucosa was noted in males (3/5) and females (2/4) and at slightly lower incidence and/or severity than the end of the dosing period and was considered to be partially recovered. Hemorrhages in the glandular mucosa were not observed. These stomach findings were consistent with a local reaction to irritation (presumptively by the test item) and, at the degree noted, were considered non-adverse.
Vacuolation of the villous lamina propria (minimal to mild) was observed in the jejunum of 9/10 males and 7/9 females at 1000 mg/kg bw/day and in 3/10 males at 300 mg/kg bw/day, and in the duodenum of 1/10 males and 3/9 females at 1000 mg/kg bw/day. This finding was characterized by discrete multiple small to large clear vacuoles in the lamina propria of the villi which most likely 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 the dosing period (no recovery). There was no associated inflammation or alteration of the tissue architecture and this finding was therefore considered to be non-adverse despite the lack of recovery. The incidence and severity of this finding was similar in males and females, therefore was considered unlikely to have contributed to the lower body weight noted in only males (end of the dosing period and recovery). For details on microscopic findings in stomach, duodenum and jejunum, please refer to Table 4 under section "Any other information on results incl. tables."
Test item-related microscopic findings were noted in the lungs of a few males and females at 1000 mg/kg bw/day and one female at 300 mg/kg bw/day. These included accumulation of material in the airways in 2/10 males at 1000 mg/kg bw/day and 1/10 females at 300 mg/kg bw/day (presumed to be test item), primarily in the large airways (bronchi and primary bronchioles), hyperplasia of the respiratory epithelium of the bronchi and bronchioles (3/10 males at 1000 mg/kg bw/day and 1/10 females at 300 mg/kg bw/day) and terminal portion of the trachea (2/10 males at 1000 mg/kg bw/day and 1/10 females at 300 mg/kg bw/day), and macrophage aggregates in the lungs (2/10 males at 100 and 300 mg/kg bw/day each and 5/10 males at 1000 mg/kg bw/day, and 2/10 females at 100 mg/kg bw/day and 1/10 females at 300 mg/kg bw/day). These macrophage aggregates of minimal severity were not considered to be test item-related as this can be observed as a spontaneous/background change in the rat. In 2/10 females dosed at 1000 mg/kg bw/day, degeneration/regeneration of the respiratory epithelium concurrent with inflammation was noted in the trachea and bronchi, with ulceration of the tracheal and/or bronchial mucosa. At 1000 mg/kg bw/day, some of the observations were considered to be adverse including accumulation of material of moderate degree with atelectasis in 1/10 males, and ulceration of the respiratory epithelium (trachea and bronchi/bronchioles) in 2/10 females. Following the recovery period, the accumulation of material was present in the air spaces in 1/5 males and 2/4 females, associated with alveolar macrophage aggregates or granulomas. These findings collectively are suggestive of gavage-related reflux and aspiration of an irritant substance, presumed to be the test item. Therefore, these are not considered as systemic effects and do not contribute to the NOAEL. Due to suspected gavage-related reflux, the nasal cavities with nasopharynx and larynx were examined from selected test item-treated animals and a few control animals. Relevant microscopic observations were only noted in two females treated at 1000 mg/kg bw/day. In one of these females, this included mild exudate and degeneration/regeneration of the respiratory epithelium of the ventral turbinates in the nasal cavity; mild ulceration, moderate degeneration/regeneration of the respiratory epithelium and moderate inflammation in the nasopharynx and marked ulceration and moderate inflammation of the larynx. In the other female, lesions were limited to the nasopharynx and included moderate degeneration/regeneration of the respiratory epithelium and minimal inflammation. For details on microscopic findings in the lungs and trachea, please refer to Table 5 under section "Any other information on results incl. tables."
In the kidney of males at 300 mg/kg bw/day an increased severity (compared to the concurrent control group) of hyaline droplet accumulation was noted (up to mild degree), and at 1000 mg/kg bw/day both increased incidence and severity were noted (also up to mild degree). The incidences were 3/10, 5/10, 6/10 and 9/10 for the control, 100, 300, and 1000 mg/kg bw/day groups. This was not accompanied by any degenerative changes and was reversible, and therefore considered non-adverse. Following the recovery period at 1000 mg/kg bw/day, the incidence and severity of this observation in 2/5 males was comparable to the control males (3/5) and therefore can be interpreted as recovered (see Table 6 under section "Any other information on results incl. tables").
In females, minor differences were noted in the amount of adipose tissue in the sternal bone marrow starting at 300 mg/kg bw/day, with minimal adipocyte accumulation noted in a 3/10 animals at 300 mg/kg bw/day and minimal to mild adipocyte accumulation noted in 5/9 animals at 1000 mg/kg bw/day. Following the recovery period, minimal adipocyte accumulation was noted in 2/4 females dosed at 1000 mg/kg bw/day, therefore considered to have partially recovered.
Minor differences in the amount of extramedullary hematopoiesis in the spleen was noted in females at 1000 mg/kg bw/day with a slightly higher incidence (5/9) and severity (up to mild) compared to the concurrent controls (2/10, mild). Following the recovery period, the single incidence of minimal extramedullary hematopoiesis was considered to be within the range of background, thus interpreted to have recovered. There was no clear hematology correlate, and these findings were therefore considered non-adverse. For details on microscopic findings in bone marrow and spleen, please refer to Table 7 under section "Any other information on results incl. tables."
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.
For details on histopathology, please refer to Attachment 1 - Table 15 under "Overall remarks, attachments".

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

ESTROUS STAGE DETERMINATION
Details on the estrous stage at the end of the dosing period can be found in Attachment 4 under "Overall remarks, attachments".

SPERM ANALYSIS
Sperm parameters were considered not affected by treatment with the test item.
The observed decrease in sperm motility and progressive sperm at 100 and 300 mg/kg bw/day was without a dose-related response and was therefore considered not test item-related. In addition, the observed increased sperm count at 1000 mg/kg bw/day was considered not test item-related as an opposite effect would be expected in case of toxicity.
For details on sperm analysis, please refer to Attachment 1 - Table 11 under "Overall remarks, attachments".

Details on results:

Abnormal breathing sounds, salivation and/or histologic changes to the lungs and/or trachea observed in one female found dead and, in several males, and females at 300 and 1000 mg/kg bw/day were suspected to be related to gastric reflux and aspiration of the test item and was considered to be a route and species-specific phenomenon, and not related to a systemic test item effect. In addition, the observed changes to hematology parameters, clinical chemistry parameters and the histopathological changes to the stomach, jejunum, duodenum, kidney, hemopoietic tissues, and changes in adrenal gland, and liver weight, in males and/or females treated at 1000 mg/kg bw/day were considered to be test item-related, but not adverse.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

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

Critical effects observed:

no

Table 1: Affected Hematology Parameters

Groups	2	3	4
Dose (mg/kg bw/day)	100	300	1000
Sex	F	F	F
White Blood Cells (10^9/L)	-	-	1.32x*
Neutrophils (10^9/L)	-	-	2.56x**
Monocytes (10^9/L)	-	-	1.99x**

F = Females
A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value at End of the Dosing period. *= P ≤ 0.05; **=P ≤ 0.01.

 

Table 2: Affected Clinical Chemistry Parameters

Groups	2		3		4
Dose (mg/kg bw/day)	100		300		1000
Sex	M	F	M	F	M	F
ALT (U/L)	-	-	-	-	1.72x**	1.36x**
AST (U/L)	-	-	-	-	1.31x**	-
Urea (mmol/L)	-	-	-	1.22x*	-	1.32x**
Cholesterol (mmol/L)	-	-	-	-	1.33x**	1.35x**
HDL cholesterol (mmol/L)	-	-	-	-	1.13x*	1.25x*
LDL cholesterol (mmol/L)	-	-	-	-	1.60x	1.48x**
Calcium (mmol/L)	-	-	1.07x*	-	1.07x	-

M = Males F = Females
A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value at End of the Dosing period. *= P ≤ 0.05; **=P ≤ 0.01.

 

Table 3: Mean percent liver and adrenal gland weight differences from control groups

	MALES				FEMALES
	Main			Recovery	Main			Recovery
Dose level (mg/kg bw/day)	100	300	1000	1000	100	300	1000	1000
BODY WEIGHT Terminal	-3	-6	-13^**	-23^**	1	-4	1	2
LIVER
Absolute	2	-2	0	-23^**	0	2	16	5
Relative to body weight	-1	4	14^**	-1	0	6	15^**	2
ADRENAL GLAND
Absolute	11	15	25^**	-14	2	3	8	7
Relative to body weight	8	20^*	42^**	11	1	7	8	5

  *: P<0.05, **: P<0.01

    Values in italics relate to dose groups that are considered test item-related

 

 

Table 4: Summary test item-related microscopic findings – stomach, duodenum and jejunum main and recovery animals

	MALES						FEMALES
	Main				Recovery		Main				Recovery
Dose level (mg/kg bw/day)	0	100	300	1000	0	1000	0	100	300	1000	0	1000
STOMACH ^a	10	10	10	10	5	5	10	10	10	9	5	4
Hyperplasia, squamous mucosa	0	0	0	7	0	3	0	0	1	6	0	2
Minimal	-	-	-	5	-	3	-	-	1	6	-	2
Mild	-	-	-	2	-	-	-	-	-	-	-	-
Hemorrhage, mucosal/submucosal, glandular	0	0	1	3	0	0	0	0	0	0	0	0
Minimal	-	-	1	3	-	-	-	-	-	-	-	-
DUODENUM ^a	10	10	9	10	5	5	10	10	10	9	5	4
Vacuolation, lamina propria, villous	0	0	0	1	0	0	0	0	0	3	0	1
Minimal	-	-	-	1	-	-	-	-	-	2	-	-
Mild	-	-	-	-	-	-	-	-	-	1	-	1
JEJUNUM ^a	10	10	10	10	5	5	10	9	10	9	5	4
Vacuolation, lamina propria, villous	0	0	3	9	0	5	0	0	0	7	0	4
Minimal	-	-	2	8	-	4	-	-	-	5	-	2
Mild	-	-	1	1	-	1	-	-	-	2	-	2

^a  =  Number of tissues examined from each group

    Values in italics relate to dose groups that are considered test item-related

 

Table 5: Summary test item-related microscopic findings – lung and rrachea main and recovery animals

	MALES						FEMALES
	Main				Recovery		Main				Recovery
Dose level (mg/kg/day)	0	100	300	1000	0	1000	0	100	300	1000	0	1000
LUNG ^a	10	10	10	10	5	5	10	10	10	9	5	4
Accumulation	0	0	0	2	0	1	0	0	1	0	0	2
Minimal	-	-	-	-	-	-	-	-	-	-	-	1
Mild	-	-	-	1	-	1	-	-	1	-	-	1
Moderate	-	-	-	1	-	-	-	-	-	-	-	-
Aggregate; alveolar, macrophage	0	2	2	5	0	1	0	2	1	0	0	3
Minimal	-	2	2	3	-	-	-	2	-	-	-	2
Mild	-	-	-	1	-	1	-	-	1	-	-	1
Moderate	-	-	-	1	-	-	-	-	-	-	-	-
Granuloma	0	0	0	1	0	1	0	0	0	0	0	0
Minimal	-	-	-	1	-	-	-	-	-	-	-	-
Mild	-	-	-	-	-	2	-	-	-	-	-	-
Hyperplasia; respiratory epithelium	0	0	0	3	0	0	0	0	1	0	0	0
Minimal	-	-	-	2	-	-	-	-	1	-	-	-
Mild	-	-	-	1	-	-	-	-		-	-	-
Atelectasis	0	0	0	1	0	0	0	0	0	0	0	0
Moderate	-	-	-	1	-	-	-	-	-	-	-	-
Degeneration/regeneration; respiratory epithelium	0	0	0	0	0	0	0	0	0	2	0	0
Mild	-	-	-	-	-	-	-	-	-	1	-	-
Moderate	-	-	-	-	-	-	-	-	-	1	-	-
Inflammation; bronchus/bronchiole	0	0	0	0	0	0	0	0	0	2	0	0
Mild	-	-	-	-	-	-	-	-	-	1	-	-
Moderate	-	-	-	-	-	-	-	-	-	1	-	-
Ulceration; bronchus	0	0	0	0	0	0	0	0	0	1	0	0
Mild	-	-	-	-	-	-	-	-	-	1	-	-
TRACHEA ^a	10	10	10	10	5	5	10	10	10	9	5	4
Hyperplasia; respiratory epithelium	0	0	0	2	0	0	0	0	1	0	0	0
Minimal	-	-	-	1	-	-	-	-	1	-	-	-
Mild	-	-	-	1	-	-	-	-		-	-	-
Inflammation	0	0	0	0	0	0	0	0	0	2	0	0
Mild	-	-	-	-	-	-	-	-	-	2	-	-
Degeneration/regeneration; respiratory epithelium	0	0	0	0	0	0	0	0	0	2	0	0
Mild	-	-	-	-	-	-	-	-	-	2	-	-
Ulceration	0	0	0	0	0	0	0	0	0	1	0	0
Mild	-	-	-	-	-	-	-	-	-	1	-	-

^a  =  Number of tissues examined from each group    

Values in italics relate to dose groups that are considered test item-related

 

 

 

Table 6: Summary test item-related microscopic findings – kidney males main and recovery animals

MALES	Main				Recovery
Dose level (mg/kg bw/day)	0	100	300	1000	0	1000
KIDNEY ^a	10	10	10	10	5	5
Accumulation, hyaline droplets	3	5	6	9	3	2
Minimal	3	5	5	5	3	2
Mild	-	-	1	4	-	-

^a  =  Number of tissues examined from each group    

Values in italics relate to dose groups that are considered test item-related

 

 

Table 7: Summary Test Item-Related Microscopic Findings – Bone marrow and Spleen Main and Recovery Animals

FEMALES	Main				Recovery
Dose level (mg/kg bw/day)	0	100	300	1000	0	1000
BONE MARROW ^a	10	10	10	9	5	4
Accumulation, adipocyte	0	0	3	5	0	2
Minimal	-	-	3	4	-	2
Mild	-	-	-	1	-	-
SPLEEN ^a	10	10	10	9	5	4
Extramedullary hematopoiesis	2	3	2	5	0	1
Minimal	2	3	2	4	-	1
Mild	-	-	-	1	-	-

^a  =  Number of tissues examined from each group    

Values in italics relate to dose groups that are considered test item-related

Conclusions:

The present study was conducted under GLP and according to OECD test guideline 408 (2018). Administration of the test substance by daily oral gavage was well tolerated in Wistar Han rats at levels up to 300 mg/kg bw/day. At 1000 mg/kg bw/day, test item-related adverse effects were observed on body weight and food consumption in males and at histopathological evaluation of the lung/trachea in males and females. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg bw/day in males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises reliable (Klimisch score 1) studies performed with the registered substance and from various source substances in the Alcohol Ethoxylates (AE) category with similar structures and intrinsic properties. Read-across is justified based on common toxicokinetic behaviour and consistent trends in environmental fate, ecotoxicological and toxicological properties of the category member substances. The data pool of the AE category is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.6, in accordance with Annex XI, Section 1.5, of the REACH Regulation (EC) No. 1907/2006.

System:

gastrointestinal tract

Organ:

stomach

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, C16-18 and C18 unsatd., ethoxylated (CAS No. 68920-66-1, EC No. 500-016-2) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Study with alcohols, C16-18 and C18 unsatd., ethoxylated (CAS No. 68920-66-1, EC No. 500-016-2)

Alcohols, C16-18 and C18-unsatd., ethoxylated (CAS 68920-66-1, EC No. 500-213-3) was administered to male and female Wistar (Crl: WI(Han) rats in a Repeated dose 90-day oral toxicity study in rodents according to OECD guideline 408 under GLP conditions (BASF, 2022a). 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.

In the high-dose group, 2/15 females were found dead on Day 51 before dosing. Abnormal breathing sounds were observed in one of the females on Days 36 - 40 and Day 43. No clinical signs were observed in the other female and no effect on body weight was observed in either of the females. For both animals, microscopic findings of neutrophilic inflammation in the surface of the heart (epicardial) and lungs (pleura) were determined. In one female, focal transmural inflammation of the esophagus, was consistent with a gavage accident as the cause of death, and therefore, not caused by the test substance. In the other female there was also accumulation of pale wispy eosinophilic material (presumed to be the test substance) in the larger airways, suggestive of gastric reflux and aspiration. Similar observations were noted in some high-dose animals in both the main and recovery groups. The irritating properties of the substance is likely to have caused the effects. Therefore, the unscheduled death of this female was considered treatment-related but not adverse, as it is related to the gavage procedure and not to intrinsic toxicological properties of the test substance.

Salivation was observed on several occasions shortly after dosing at all dose levels (except low-dose females) with a dose-related incidence. 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 in 0/15, 1/10, 1/10 and 6/15 males in the control, low- mid- and high-dose group, respectively, and in 0/15, 0/10, 1/10 and 4/15 females in the control, low- mid- and high-dose group, respectively, during the dosing period. During the recovery period, 1/5 high-dose males had abnormal breathing sounds once. Based on the 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.

In mid-dose males a lower body weight gain compared to controls between Days 36 and 43 was noted, which resulted in a 6.8% lower body weight compared to the controls at the end of the dosing period (not statistically significant). High-dose males showed lower body weight gain from Day 22 onwards during the dosing period, compared to the controls. This resulted in a 15.5% lower body weight compared to controls at the end of the dosing period. Throughout the recovery period, the body weight in high-dose males remained decreased compared to controls (-22% at end of recovery period). This was considered to be an adverse effect. No treatment-related effects were observed in females.

Mid- and high-dose males showed a slightly lower food consumption compared to controls throughout the dosing period, resulting in a 5.5% and 6.9% lower food consumption during the complete dosing period (Day 1 - 91), respectively, compared to controls. The food consumption in high-dose males remained lower throughout the recovery period (-21% at end of recovery period, compared to controls).

High-dose females showed increased white blood cell (1.32 x, p ≤ 0.05), neutrophil (2.56 x, p ≤ 0.01), and monocyte (1.99 x, p ≤ 0.01) counts at the end of the dosing period, compared to controls. These changes were no longer present at the end of the recovery period. As there was no microscopic correlate and the magnitude of change observed was limited, these findings were considered not adverse.

Changes in clinical chemistry parameters at the end of the dosing period included an increase in aspartate aminotransferase (AST) activity in high-dose males and females (1.72 and 1.36 x of control, respectively, statistically significant), and an increase in alkaline phosphatase (ALP) activity in high-dose males (1.31 x, statistically significant), compared to controls. An increase in cholesterol level was observed in high-dose males and females (1.33 x and 1.35 x, respectively; statistically significant) compared to controls. A similar increase was noted in HDL cholesterol levels (1.13 x (males) and 1.25 x (females); statistically significant) and in LDL cholesterol levels (1.60 x (males) and 1.48 x (females), statistically significant in females only) at the high-dose level, compared to controls. In the mid-dose females increased urea concentrations (1.22 x, statistically significant) were noted, while slightly increased calcium concentrations (1.07 x, statistically significant) were observed in mid-dose males at end of the dosing period, compared to controls. These changes were no longer present at end of the recovery period. As there was no microscopic correlate and the magnitude of change observed was small, these findings were considered not adverse.

At the end of the dosing period thyroid stimulating hormone (TSH) concentrations were increased in high-dose males (1.52 x), and in females at all dose levels (2.59 x, 1.53 x and 1.95 x by increasing dose, respectively) compared to controls. At end of the recovery period, TSH concentrations were reduced in males, compared to controls. In females, TSH concentrations were increased compared to the end of the dosing period (4.26 x, dose level not reported). As all mean values remained within the historical control data range and lacked a clear dose-response, these results were considered unrelated to treatment with the test substance. Hormonal levels of T3 and T4 were unaffected by treatment, thereby supporting a lack of relevant effects on thyroid homeostasis.

The observed decrease in sperm motility and progressive sperm in males in the low- and mid-dose groups did not show a dose-related response and was therefore considered incidental and not treatment-related. As the sperm count change in high-dose males was an increase, this was not considered adverse.

In the main group, increased liver weights were noted in males and females in the high-dose group. The increases were statistically significant relative to body weight in males (14%) and females (15%), compared with the control. Following the recovery period the relative 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 males in the mid-and high-dose levels, compared with the control. The increase relative to body weight was statistically significant in mid-and high-dose males (20 and 42%, respectively) compared with control, while for the absolute weight a statistically significant increase (25%) was noted high-dose males only. The adrenal weight was similar in all groups at the end of the recovery period, and thus, the effect on adrenal weight is not considered to be toxicologically relevant.

Treatment-related macroscopic findings were recorded in the high-dose group at the end of the dosing period in the stomach. Dark red foci in the glandular mucosa of the stomach (correlating to acute mucosal haemorrhage) was noted in 0/10, 0/10, 1/10 and 4/10 males in the control, low- mid- and high-dose group, respectively.

Microscopic findings were noted in the duodenum, jejunum, stomach and lungs of males and females, and in the kidney of males, and in the spleen and bone marrow of females.

In the non-glandular stomach squamous mucosal hyperplasia (minimal to mild) was observed in 0/10, 0/10, 0/10 and 7/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 1/10 and 6/10 females in the control, low- mid- and high-dose group, respectively, following the dosing period. Following the recovery period, squamous mucosal hyperplasia was noted in 0/5 and 3/5 males in the control and high-dose group, respectively, and in 0/5 and 2/5 females in the control and high-dose group, respectively. In the glandular stomach, minimal 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. No cases were noted after the recovery period. The few occurrences in the low-, and mid-dose groups were not considered to be clear indications of a treatment-related effect. Due to the limited effect on the non- glandular stomach, this is not considered to be an adverse effect.

Vacuolation of the villous lamina propria of the jejunum was observed following the dosing period in 0/10, 0/10, 3/10 and 9/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 7/10 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. Vacuolation of the villous lamina propria was also observed in the duodenum, although in lower numbers than those for the jejunum (1/10 high-dose males and 3/10 high-dose females). 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 3/10, 5/10, 6/10 and 9/10 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 minimal to mild accumulation. Following the recovery period 3/5 and 2/5 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.

Test item-related microscopic findings were noted in the lungs of 1/10-5/10 high-dose males and 1/10-2/10 high-dose females, as well as in 1/10 mid-dose females. These included accumulation of material in the airways in 2/10 high-dose males and 1/10 mid-dose females (presumed to be test substance), primarily in the large airways (bronchi and primary bronchioles); and hyperplasia of the respiratory epithelium of the bronchi and bronchioles (3/10 high-dose males and 1/10 mid-dose females) and terminal portion of the trachea (2/10 high-dose males and 1/10 mid-dose females).

Macrophage aggregates were noted in the lungs in 0/10, 2/10, 2/10 and 5/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 2/10, 1/10 and 0/10 females in the control, low- mid- and high-dose group, respectively. These macrophage aggregates of minimal severity were not considered to be test item-related as this can be observed as a spontaneous/background change in the rat. In 2/10 high-dose females, degeneration/regeneration of the respiratory epithelium concurrent with inflammation was noted in the trachea and bronchi, with ulceration of the tracheal and/or bronchial mucosa. In the high-dose group, some of the observations were considered to be adverse, including accumulation of material of moderate degree with atelectasis in 1/10 males, and ulceration of the respiratory epithelium (trachea and bronchi/bronchioles) in 2/10 females. Following the recovery period, the accumulation of material was present in the air spaces in 1/5 high-dose males and 2/4 high-dose females, associated with alveolar macrophage aggregates or granulomas. These findings collectively are suggestive of gavage-related reflux and aspiration of an irritant substance, presumed to be the test item.

In females, minor differences were noted in the amount of adipose tissue in the sternal bone marrow starting at the mid-dose, with minimal adipocyte accumulation noted in 3/10 mid-dose females and minimal to mild adipocyte accumulation noted in 5/9 high-dose females. Following the recovery period, minimal adipocyte accumulation was noted in 2/4 high-dose females, therefore considered to have partially recovered and not to be an adverse effect.

Minor differences in the amount of extramedullary haematopoiesis in the spleen was noted in high-dose females with a slightly higher incidence (5/9) and severity (up to mild) compared to the concurrent controls (2/10, mild). Following the recovery period, the single incidence of minimal extramedullary haematopoiesis was considered to be within the range of background numbers. There was no clear haematology correlate, and these findings were therefore considered non-adverse.

The results of the 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) = 300 mg/kg bw/day for systemic toxicity in males and ≥ 1000 mg/kg bw/day for systemic toxicity in females 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

Evaluation of repeated dose toxicity as observed in available studies

In the AE category, the database for subacute RDT consists of eight combined repeated dose toxicity studies with the reproduction / developmental toxicity screening test. The studies were performed with five substances in the ‘linear’ and three in the ‘mixed branched & linear’ subgroup of the category. The database for subchronic RDT contains four subchronic (90-day) RDT studies conducted with four different AE substances: two in the ‘linear’ and two in the ‘mixed branched & linear’ subgroup, respectively.

The combined repeated dose toxicity study with the reproductive / developmental toxicity screening test was performed according to OECD guideline 422 under GLP conditions. Groups of 10 rats per sex received the test substance 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. The dose levels were set based on the guideline recommendation for substances not expected to exhibit strong systemic toxicity. Males were treated for 29 days whereas females that delivered were treated for 50-64 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females that failed to deliver or had a total litter loss were treated for 40-43 days. The following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations. In addition, a number of reproduction / developmental parameters were investigated.

The Repeated dose 90-day oral toxicity study in rodents was performed according to OECD guideline 408 under GLP conditions. Groups of 10 rats/sex were administered the test substance 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 800 mg/kg bw/day group to assess the recovery from any treatment-related effects. The dose levels were based on the results of the OECD 422 studies. 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.

The dose levels applied in almost all the main studies were 100, 300 and 1000 mg/kg bw/day in both the subacute and subchronic studies (95, 285 and 950 mg/kg bw/day in the subacute study with decan-1-ol, ethoxylated, CAS No. 26183-52-8). The recommended limit dose of the test guidelines was chosen as the highest dose. In the subchronic study with alcohols, C9-11, branched and linear, ethoxylated dose levels of 100, 300 and 800 mg/kg bw/day were used as the top dose of 1000 mg/kg bw/day was considered to cause effects too severe for a 90-day study due to the irritating properties of this substance.

Certain effects were noted in several of the studies, although the effects were not always considered to be adverse or toxicologically relevant. All of the effects that were considered adverse were observed in several or most of the studies, indicating the same target organs and tissues were affected by different AE substances. As can be expected for surfactants with known irritating properties, some effects caused by irritation at the site of contact (fore-stomach) were observed in the studies. Only parameters relevant for RDT (i.e. only the parental parameters from the OECD 422 studies) are summarised in this section. The full assessment of the reproductive and developmental toxicity parameters investigated in the OECD 422 studies is provided in the endpoint summary for IUCLID section 7.8. The following observations were considered to be characteristic to the AE substances.

 

Clinical signs

Several clinical signs were observed consistently in several or most of the subacute and subchronic studies. Salivation was noted in most studies, with numbers increasing with dose level. This was considered a physiological response rather than a sign of systemic toxicity. A flat and/or hunched posture, increasing in number with the dose, was noted particularly in the subacute studies. Abnormal breathing sounds and piloerection were observed particularly in high-dose animals in some of the subacute- and all the subchronic studies. In the subchronic studies, no or a greatly reduced occurrence of clinical signs were noted during the recovery period. Based on the incidence and severity as well as due to the persistent and recurrent nature, the clinical signs were considered an adverse effect in OECD 422 studies with alcohols, C9-11, branched and linear, ethoxylated and alcohols, C12-13, branched and linear, ethoxylated. In all the other RDT studies (both subacute and subchronic), the clinical signs were considered to be a non-adverse response to the treatment.

 

Body weight development

A lower body weight gain of mid- and high-dose males, compared with the control, was observed in most of the available RDT studies. The reduced body weight gain was below 10% compared to controls at most time points but reached 15.5% for high-dose males in the subchronic study with alcohols, C16-18 and C18-unsatd., ethoxylated. In this study, the lower body weight gain, compared with the control, was considered adverse. A slightly increased body weight development was apparent for treated females compared with the control in some studies. The reduced body weight development could be caused by the irritating properties of the AE substances and the local effects found in the (fore)stomach. An irritated and locally damaged stomach generally lead to a reduced food consumption as observed in most of the RDT studies (both, subacute and subchronic). The reduction in food intake is reflected by a slightly lower body weight development when compared to control animals.

 

Stomach

Macroscopic and microscopic alterations in the stomach and/or forestomach were observed in 4/8 subacute RDT studies (with alcohols, C12-14, ethoxylated; hexadecan-1-ol, ethoxylated; alcohols, C9-11, branched and linear, ethoxylated; and alcohols, C12-15, branched and linear, ethoxylated) and in 4/4 subchronic RDT studies (with alcohols, C12-14, ethoxylated; alcohols, C16-18 and C18-unsatd., ethoxylated; alcohols, C9-11, branched and linear, ethoxylated; and alcohols, C12-15, branched and linear, ethoxylated). Although the effects were found in animals of both sexes and at all dose levels, they were most prominent and severe in high-dose males. Macroscopic effects included dark red foci in the glandular mucosa and an irregular surface of the non-glandular stomach. During the microscopic examination squamous mucosal hyperplasia (correlated to irregular surface) with hyperkeratosis (in some cases accompanied by (sub)mucosal (lympho)granulocytic infiltrate), oedema and ulceration (most often at the limiting ridge) were found in the non-glandular stomach. In the glandular mucosa, focal acute mucosal haemorrhage occurred (correlated to dark red foci). The animals only partially recovered from these effects in the satellite group of the subchronic studies. Based on the incidence and severity, the findings in the non-glandular region of the stomach (forestomach) were considered adverse in 1/8 subacute studies (with hexadecan-1-ol, ethoxylated) and in 2/4 subchronic studies (with alcohols, C9-11, branched and linear, ethoxylated and alcohols, C12-15, branched and linear, ethoxylated). They were generally assessed to be local effects due to the irritating properties of the tested AE substances. However, since humans lack a forestomach, they are not relevant to assess human health hazards of the substances. An important observation is that none of the effects found in the glandular region of the stomach (i.e. that might be relevant for humans) were considered adverse.

Gastrointestinal tract, jejunum

In 4/8 subacute studies (with alcohols, C12-14, ethoxylated; hexadecan-1-ol, ethoxylated; (Z)-9-Octadecen-1-ol ethoxylated; and alcohols, C12-15, branched and linear, ethoxylated) and in 3/4 subchronic studies (with alcohols, C12-14, ethoxylated; alcohols, C16-18 and C18-unsatd., ethoxylated; and alcohols, C12-15, branched and linear, ethoxylated) histological changes in the jejunum were observed for mid- and high-dose males and females. These changes consisted of multifocal villous vacuolation characterised by variable sized clear vacuoles in the lamina propria of the villi. The vacuoles occasionally contained a minimal amount of lacy flocculent eosinophilic to amphophilic material. In some vacuoles, lining of attenuated endothelial-like cells was present. It is most likely that the vacuoles represent dilated lacteals. The effects were still observed at the end of the recovery period in the subchronic studies, demonstrating no recovery in both sexes. However, as it did not appear to affect the health or digestion of the animals, it was not considered a toxicologically relevant effect.

 

Liver

Effects on the liver were found in animals of both sexes with increasing incidence and severity by increasing doses, starting at the low-dose level, in 5/8 subacute studies (with decan-1-ol, ethoxylated; alcohols, C12-14, ethoxylated; alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO; alcohols, C9-11, branched and linear, ethoxylated; alcohols, C12-13, branched and linear, ethoxylated and alcohols, C12-15, branched and linear, ethoxylated) and 3/4 subchronic studies (with alcohols, C12-14, ethoxylated; alcohols, C9-11, branched and linear, ethoxylated and alcohols, C12-15, branched and linear, ethoxylated). Effects observed included prominent lobular architecture and pale or pale tan discoloration. While the discoloration was found without microscopic correlate, the prominent lobular architecture correlated to centrilobular hepatocellular hypertrophy and increased liver weights (up to +55% absolute) in the subchronic study with alcohols, C12-14, ethoxylated. Higher mean liver weights in the treated groups than in the control groups were found in all RDT studies (statistically significant for absolute and/or relative weight in all high-dose groups and in some low- and mid-dose groups). The observations are considered adaptive changes as a reaction to an increased metabolic burden due to gavage administration of substantial amounts of test material in corn oil. Administration of corn oil alone can be expected to result in increased fat / oil metabolism. Therefore, the effects in all studies were evaluated as non-adverse. This assessment is further supported by the fact that the analysis of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) - although increased in some studies - did not indicate the presence of liver damage.

 

Lungs and airways

In the subchronic RDT study with alcohols, C16-18 and C18-unsatd., ethoxylated accumulation of material was found in the airways (primarily in large airways like bronchi and primary bronchioles) of mid- and high-dose animals. The material was presumably the test substance. This finding was accompanied by hyperplasia of the respiratory epithelium (bronchi, bronchioles and terminal portion of trachea), macrophage aggregates, degeneration/regeneration of respiratory epithelium concurrent with inflammation in the trachea and bronchi (with ulceration of tracheal and/or bronchial mucosa). These findings collectively were evaluated to be suggestive of gavage-related reflux and aspiration of the irritant test substance. No effects on the lungs and respiratory system were noted in any other studies.

 

Sperm analysis

A decreased percentage progressive sperm and/or a decreased motility was observed in males of all dose levels in 3/4 subchronic studies (with alcohols, C16-18 and C18-unsatd., ethoxylated; alcohols, C9-11, branched and linear, ethoxylated and alcohols, C12-15, branched and linear, ethoxylated). The findings were considered adverse only for alcohols, C9-11, branched and linear, ethoxylated. In addition, an increased number of cells with other tails was noted in high-dose males in the subchronic study with alcohols, C12-15, branched and linear, ethoxylated. Sperm analysis was not performed in the OECD 422 studies. However, there are no indications of effects on sperm quality in the OECD 422 studies as parameters such as mating and fertility indices and pre-coital time were completely unaffected.

 

Oestrous cycle

Most females had regular cycles of 4 to 5 days and extended di-oestrous occurred in a few females only. However, in the OECD 422 study with alcohols, C9-11, branched and linear, ethoxylated, 3/10 high-dose females were acyclic, one had an irregular cycle and for one female it was not possible to determine the length and regularity of the oestrous cycle. As 5/10 females had a disturbed cycle this was considered an adverse finding in that study. None of the observations on oestrus cycle made in the seven other OECD 422 studies was considered to be adverse. Moreover, no effects on oestrus cycle were found in any of the subchronic studies.

In addition to the above-listed observations, some results not considered to be specific to treatment with the AE substances were reported. Several animals died during the dosing period in both subacute and subchronic studies. These cases were not treatment-related and/or not toxicologically relevant. No clear picture with respect to effects on haematology, clinical chemistry and thyroid hormone levels could be derived from the RDT studies. Some values were increased for some substances but decreased for others. Sometimes values were increased in one dose level and decreased in another in the same study. Therefore, these observations were considered to reflect the natural variation in animals exposed to xenobiotics over an extended period of time and kept under laboratory conditions. They are not indicative of a specific systemic toxicity of the investigated AE substances. Observed increases in kidney weight for males and/or females in some RDT studies was considered non-adverse as the effect was not observed following the recovery period. Hyaline droplet accumulation in the kidney of male rats was noted in 3/4 subchronic studies (with alcohols, C12-14, ethoxylated; alcohols, C16-18 and C18-unsatd., ethoxylated and alcohols, C12-15, branched and linear, ethoxylated). This effect is well-known in male rats under similar treatment condition and is not considered relevant to humans.

Conclusion on RDT

In the RDT studies, effects or changes that appeared characteristic of the AE substances were observed: The increased liver weight and changes in liver histopathology were noted in most of the subacute and subchronic studies, and were most likely adaptive changes as a reaction to an increased metabolic burden due to gavage administration of substantial amounts of test substance. A reduced body weight (gain) observed in most of the subacute and subchronic studies was considered a result of general discomfort in rats administered the test substance. The specific cause of the jejunal effects, which were considered non-adverse, could not be identified. The changes in the non-glandular stomach and glandular stomach were caused by the irritant effect of the AE substances at the site of contact, and therefore considered a local effect. No Mode of Action was identified, which is consistent with the proposed toxicokinetic behaviour of AE substances (refer to the endpoint summary for section 7.1). Hydrolysis is an important step of the metabolic breakdown, which takes place either in the gastro-intestinal tract or in the liver after absorption of the parent compounds; resulting in free alcohols and (oligo-)ethylene glycols (refer to discussion of underlying mechanism). The subsequent metabolism of both primary metabolites is well known (e.g. oxidation) and is not expected to result in metabolites exhibiting significant toxicity. The unspecific systemic effects observed are therefore the result of a generally low systemic toxicity caused by the AE substances. Due primarily to the increased severity of clinical signs caused by the ‘mixed branched & linear’ AE substances, No-Observed-Adverse-Effect-Levels (NOAELs) are generally lower than those for ‘linear’ AE substances (see Table 1). Lower NOAELs are also derived for most of the subchronic studies, compared with the subacute study of the relevant substances.

The data available for alcohols, C16-18 and C18 unsatd., ethoxylated (CAS No. 68920-66-1, EC No. 500-016-2) is consistent with the overall RDT data for AE substances. The following NOAELs were set:

 

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) = 300 mg/kg bw/day

 

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

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

 

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, C16-18 and C18 unsatd., ethoxylated (CAS No. 68920-66-1, EC No. 500-016-2) 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.