Alcohols, C9-11, branched and linear, ethoxylated

Endpoint summary

Description of key information

Oral (subacute, rat, m/f, OECD 422): NOAEL (systemic toxicity) = 300 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) = 300 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, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) 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:

23 Jan - 12 Jun 2020

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

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. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.

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: 11-13 weeks
- Weight at study initiation: 205-242 g
- Fasting period before study: NA
- 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: 8 days for males and 10 days for females

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): 20-22
- Humidity (%): 35-53
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 23 JAN 2020 to 30 MAR 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected following a Compliance Check Final Decision (CCH-D-2114493190-51-01/F). Since the registered substance is a liquid, ECHA concluded that testing should be performed by the oral route.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- 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.
- Concentration in vehicle: 5 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.
- 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 ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20219836).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver 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 Finder with oral administration of Alcohols, C9-11, branched and linear, ethoxylated in rats and in an attempt to produce graded responses to the test item (Test Facility Study No. 20219837).
- 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.

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 NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

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, c, harderian, lacrimal, mammary, parathyroid, 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.

Other examinations:

Measurement of total T4 was conducted for F0-males. Assessment of T4 in F0-females and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males.

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 was 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 is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For all females treated at 1000 mg/kg/day, piloerection and slight to severe lethargy were noted during the entire study period. In the beginning of the study, the majority of the animals were affected and this was accompanied by abnormal (7/10), flat (9/10) and hunched posture (10/10) and/or uncoordinated movements (9/10). At the end of study, these clinical signs were noted in individual animals and (ventro-)lateral recumbency (1/10), loss of righting reflex (1/10), decreased locomotor activity (2/10) and/or abnormal gait (1/10) were also noted in individual animals. Rales (6/10) was noted for the majority of females during the entire study period, which was incidentally accompanied with slow breathing (2/10), labored (1/10), deep (1/10) and/or shallow respiration (2/10) for individual animals. Other clinical signs that were incidentally noted for individual animals included chromodacryorrhoea (1/10), lean appearance (2/10) and ptosis (3/10). For males treated at 1000 mg/kg/day, rales (8/10) was noted for the majority of the animals during the last two weeks of the study, which was accompanied by labored respiration (2/10) in individual animals. Clinical signs noted in a majority of the animals were considered toxicologically relevant. Piloerection (1/10) was also incidentally noted for individual animals. At the incidence observed, these clinical signs were considered not toxicologically relevant. At 300 mg/kg/day, rales and chromodacryorrhoea were incidentally noted in males and piloerection was incidentally noted in females, based on the incidence observed, these clinical signs were considered not toxicologically relevant. Salivation seen after dosing for males and females at 1000 mg/kg/day and males at 300 mg/kg/day 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 related to taste of the test item rather than a sign of systemic toxicity. No clinical signs were noted for the control, animals treated at 100 mg/kg/day and during weekly arena observations.

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality was noted up to 1000 mg/kg/day.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were observed up to 1000 mg/kg/day. A slightly lower body weight gain was noted for males treated at 1000 mg/kg/day during the first week of treatment. At the end of the study period, absolute body weight was of 0.97x of controls. Based on the magnitude of the change this was considered not toxicologically relevant. No effect on body weight or body weight gain was noted for females treated up to 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were noted up to 1000 mg/kg/day. Relative food consumption during the lactation phase was slightly lower for females treated at 1000 mg/kg/day (up to 0.92x of control; not statistically significant). Based on the magnitude of the change this was considered not toxicologically relevant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item. Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item. The longer prothrombin time (PT) for males treated at 100 mg/kg/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were noted in clinical biochemistry parameters after treatment with the test item up to 300 mg/kg/day. For females treated at 1000 mg/kg/day, an increased bile acids concentration was noted (2.53x of control), which was slightly outside the range of historical control data. Furthermore, a decreased creatinine concentration was noted (0.89x of control), which was also slightly outside the range of historical control data. Based on the magnitude of the
change and as an opposite effect would be expected in case of target organ toxicity, the effect on creatinine was considered not toxicologically relevant. For males treated at 1000 mg/kg/day, inorganic phosphate concentrations were increased (1.18x of control), based on the magnitude of the change and as mean values were within historical control data, this was considered not toxicologically relevant. The increased potassium concentration for males of all treatment groups was considered to have arisen as a result of slightly low control values and not related to treatment with the test item. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment with the test item up to 1000 mg/kg/day. A higher hind limb grip strength was noted for males treated at 1000 mg/kg/day, which was considered to have arisen as a result of slightly low control values. Furthermore, a decreased grip strength would be expected in case of target organ toxicity. Therefore, this change was considered not related to treatment with the test item. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg/day. Motor activity was similar between 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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver weights (absolute and relative to body weights) were noted in the 300 and 1000 mg/kg/day groups males, as shown in Table 1. Any other differences were considered not to be Alcohols, C9-11, branched and linear, ethoxylated-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values. The higher liver weight observed in the 300 and 1000 mg/kg/day group males was without microscopic correlation and without test item-related changes of liver enzymes, therefore it was considered non-adverse.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were present in the forestomach of two 1000 mg/kg/day rats of each sex, in the form of irregular surface, combined with a thickened limiting ridge in one female. The microscopic correlates of these two macroscopic findings consisted of squamous cell hyperplasia with hyperkeratosis in both sexes and/or edema in males. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse.
Furthermore, a reduced size of the thyroid gland was noted in two males and one female treated at 1000 mg/kg/day. In the males, this finding was unilateral observed. There was no microscopic correlation or test-item related effect on the mean thyroid gland weight. In both sexes, the individual absolute weight and relative to body weight ratios of the thyroid glands of the animals with the macroscopic finding of reduced size, were higher than the lowest concurrent control values. Therefore, this macroscopic finding was regarded as unrelated to treatment with the test item and 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.

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 forestomach of both sexes and thyroid gland of males of the 1000 mg/kg/day group and are summarized in Table 2. At 1000 mg/kg/day, signs of local irritation of the forestomach (i.e. non-glandular stomach) were present in the form of squamous cell hyperplasia with hyperkeratosis and subepidermal lymphogranulocytic infiltrate in both sexes and additional sub(mucosal) edema in males. The macroscopic correlate for the hyperplasia and edema consisted of irregular surface of the forestomach and/or thickened limiting ridge. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse. The test item-related increased incidence of follicular cell hypertrophy of the thyroid gland of 1000 mg/kg/day males was at the observed minimal severity and in absence of other test-item related thyroid gland changes regarded non-adverse.
The remainder of the recorded microscopic findings 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. This included the requested liver of males, showing incidences of one control, one 100 mg/kg/day, zero 300 mg/kg/day and two 1000 mg/kg/day group males with minimal hepatocellular hypertrophy.

Histopathological findings: neoplastic:

not examined

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:

clinical signs

Key result

Critical effects observed:

no

Table 1
Mean Percent Liver Weight Differences from Control Groups

	Males
Dose level (mg/kg/day):	100	300	1000
LIVER
Absolute	1	15	15
Relative to body weight	-2	11*	19**

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

Table 2
Summary Test Item-Related Microscopic Findings – Forestomach and Thyroid Gland

	Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia, with hyperkeratosis
Minimal	-	-	-	2	-	-	-	1
Slight	-	-	-	2	-	-	-	2
Edema
Minimal	-	-	-	1	-	-	-	-
Slight	-	-	-	1	-	-	-	-
Infiltrate, lymphogranulocytic
Minimal	-	-	-	3	-	-	-	1
THYROID GLAND ^a	5	5	5	6	6	0	0	5
Follicular cell hypertrophy
Minimal	1	-	1	3	-	n.a.	n.a.	-
Slight	-	1	-	1	-	n.a.	n.a.	-

^a  =  Number of tissues examined from each group; n.a.= not applicable

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Mar - 19 Oct 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:

- rel. humidity outside range (max 74%) for 7 days (minimal change, not a major deviation) - 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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 170 - 212 g (males) and 108 - 152 g (females)
- Housing: Up to 5 animals of the same sex and same 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, DE) equipped with water bottles. 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 diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, DE); ad libitum (except during motor activity measurements)
- Water: municipal tap water; ad libitum (except during motor activity measurements)
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants, as well as periodic analysis of water were routinely performed. No known contaminants in the feed that would interfere with the objectives of the study were present.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 47 - 74
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 15 Apr - 16 Aug 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared weekly and stored at 2 - 8 °C. Formulations (w/w) were homogenized to visually acceptable levels by stirring continuously during dosing. An adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item. The dosing volume was 3 mL/kg bw, 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 analyses; trial preparations were performed and showed suitability of the vehicle
- Concentration in vehicle: 33.3, 100 and 266.7 mg/mL for 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:

Formulation analyses confirmed that formulations of the test item in corn oil were prepared accurately and homogenously. Regarding accuracy, the concentrations analyzed in the formulations of Groups 2, 3 and 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 85 - 115% of target concentration). A small response at the retention time of the ammonium adduct of C10E6 was observed in the chromatograms of the Group 1 formulations prepared for use in Weeks 1, 6 and 12. The maximum contribution to the Group 2 samples was 0.010% and therefore this small response was considered negligible and not to derive from the test item.
As regards 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.:

800 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 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

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 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 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 to determine the stage of estrous.
- Sperm analysis: from all surviving males; evaluated for sperm 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 will be 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):

Main study:
Abnormal breathing sounds were noted in 3/10 males and 2/10 females at 300 mg/kg bw/day on some occasions between Days 3 and 75, and in 13/15 males and 10/15 females at 800 mg/kg bw/day on many occasions throughout the dosing period (more pronounced in males). Labored breathing was observed for 1 male at 300 and 800 mg/kg bw/day each on one or two days, and shallow breathing occurred in 1/15 males and females at 800 mg/kg bw/day on Day 5 only.
Prostration was observed for 4/15 males and 9/15 females at 800 mg/kg bw/day on several occasions between Days 42 and 91. In addition, decreased activity was noted for 4/15 males and 10/15 females at 800 mg/kg bw/day on several occasions during the dosing period; for 1 male, this was accompanied by an abnormal gait on Day 79. Moreover, a hunched posture was noted on several occasions for 5/15 males and 4/15 females at 800 mg/kg bw/day. Finally, erected fur was seen for 6/15 males and 5/15 females at 800 mg/kg bw/day on some occasions.
Salivation was observed after dosing in males and females starting at 100 mg/kg bw/day (7/10 males and 6/10 females) with a dose-related increase in incidence and severity (all males at 300 mg/kg bw/day and 1000 mg/kg bw/day, and 8/15 females at 300 mg/kg bw/day and all females at 1000 mg/kg bw/day). Additionally, it was also seen in 1/15 control female on 1 occasion. This 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.
No findings were noted during the arena observations in this study.
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. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Recovery groups:
Abnormal breathing sounds were noted once for 1/5 male and on several occasions for 2/5 females at 800 mg/kg bw/day. No other signs of note were recorded during the 28-day treatment-free period.
For details on clinical signs, please refer to Attachment 1 - Table 1 under section "Attached background material".

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.
Two females at 300 mg/kg bw/day were found dead after dosing on Day 8 (No. 79) or 13 (No. 84). No signs were observed prior to their death. At necropsy, clear, watery fluid was noted in the thoracic cavity of both females. In addition, dark red discoloration of the left lung and dark red foci on the thymus were noted for Female No. 79 and for Female No. 84, the lungs failed to collapse. All macroscopic findings were without microscopic correlate and there were no microscopic findings of note that could explain the cause of death of these animals (i.e., no evidence of gavage accident or gavage-related reflux). These premature deaths were considered to have resulted from an incident unrelated to the treatment with the test item, given the lack of a dose response and in the absence of any other supporting findings.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Main study:
Body weights and body weight gain of males up to 300 mg/kg bw/day and females up to 800 mg/kg bw/day remained in the same range as controls over the study period. Body weights and body weight gain in males at 800 mg/kg bw/day were slightly lower when compared to control. Body weight gain was statistically significantly decreased over Days 1 - 8 (39.7 g), Days 8 - 15 (33.1 g) and 43 - 50 (9.8 g) of the dosing period only, resulting in an overall weight gain of 0.91x of control and a mean body weight of 0.93x of control at the end of the dosing period (not statistically significant).
Body weight gain during the recovery period was considered to be similar to controls. The slightly increased weight gain for males at 800 mg/kg bw/day over Days 106 - 113 was considered to be a chance finding.
All these changes in body weight and body weight gain were considered non-adverse.
For details on body weight and body weight gains, please refer to Attachment 1 - Figure 1 and Tables 2 - 3 under section "Attached background material."

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Main study:
Food consumption of males up to 300 mg/kg bw/day was similar to the control level over the study period. Food consumption was slightly lower in males at 800 mg/kg bw/day on several occasions during the dosing period (up to 0.92 x of control; not statistically significant). For females at all dose levels, food consumption was slightly lower over Days 1 - 8 (0.93, 0.92 and 0.91x at 100, 300 and 800 mg/kg bw/day, respectively; not statistically significant), and for females at 100 mg/kg bw/day over Days 8 - 15 (0.92 x; not statistically significant). As the food consumption was only decreased in the first two weeks of the study and was comparable with controls during the remainder of the Dosing Period, this finding was considered to be not toxicologically relevant.
Recovery group:
During the recovery period, food consumption was in general similar to control.
For details on food consumption, please refer to Attachment 1 - Figure 2 and Tables 4 under section "Attached background material."

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmology findings were noted that were considered to be related to treatment with the test item. 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. The findings at Week 13 were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of males up to 300 mg/kg bw/day and females up to 800 mg/kg bw/day were considered not to have been affected by treatment with the test item.
In males at 800 mg/kg bw/day, red blood cell distribution width (RDWG) was decreased at the end of the dosing period (0.96 x of control), reaching statistical significance. At the end of the recovery period, monocyte (MONO) count and hemoglobin (HGB) concentration were statistically significantly decreased in males at 800 mg/kg bw/day (0.70 and 0.95 x, respectively).
All other values were similar to control. Platelet clumps were seen in one control female (No. 58) at the end of the dosing period. As this finding occurred in a single control animal, this was not test item-related. 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, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
For details on haematology, please refer to Attachment 1 - Table 7 under section "Attached background material".
Coagulation:
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item.
For details on coagulation, please refer to Attachment 1 - Table 8 under section "Attached background material".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of males up to 300 and females up to 800 mg/kg bw/day were considered not to have been affected by treatment with the test item. Decreased triglycerides (TRIG; 0.76 x of control, not statistically significant) and increased potassium (K; 1.09 x, statistically significant) concentrations were noted in males at 800 mg/kg bw/day at the end of the dosing period.
At the end of the recovery period, all values were considered to be similar to control. Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to the test item as these occurred in the absence of a dose-related trend.
For details on clinical biochemistry, please refer to Attachment 1 - Table 9 under section "Attached background material".

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid stimulating hormone (TSH) levels were increased in males at 300 and 800 mg/kg bw/day (1.40 and 1.27 x of control, respectively) and in females at all dose levels (2.17, 1.45 and 2.25 x, respectively) at the end of the dosing period, albeit without reaching statistical significance. In addition, Thyroxine (T4) levels were increased in females at 800 mg/kg bw/day (1.22 x, not statistically significant).
At the end of the recovery period, the mean TSH level was decreased in males at 800 mg/kg bw/day (0.43 x; not statistically significant) but increased in females (1.44 x; not statistically significant). In addition, Triiodothyronine (T3) concentration was increased in females at 800 mg/kg bw/day (1.23 x; statistically significant) after the 28-day treatment-free period. All mean values at the end of treatment and recovery period were well within the historical control range considered normal for rats of this age and strain. Any other statistically significant changes in thyroid hormone parameters were considered to be unrelated to the test item as these occurred in the absence of a dose-related trend.
For details on thyroid hormones, please refer to Attachment 1 - Table 9 under section "Attached background material".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine parameters (pH and volume) of males up to 300 mg/kg bw/day and females up to 800 mg/kg bw/day were considered not to have been affected by treatment with the test item. The urinary pH was decreased in males at 800 mg/kg bw/day at the end of the dosing period (0.90 x of control, statistically significant). At the end of the recovery period, all values were similar to control.
For details urinalysis, please refer to Attachment 1 - Table 10 under section "Attached background material".

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were similar in all examined animals. Grip strength was considered to be similar between control and high dose animals. The slightly higher grip strength of the hindlegs in females at 300 and 800 mg/kg bw/day (1.31 and 1.30 x of control, both statistically significant) was considered to be unrelated to treatment with the test item, given the direction of change (increase) and in the absence of a clear dose-related response.
Motor activity was considered to be similar between treated and control groups. For males at 800 mg/kg bw/day, the mean total movements were lower compared to control (0.67 x of control, statistically significant). This was considered to be the result of relatively high control values and as all values remained well within the range considered normal for rats of this age and strain, this difference was considered to be unrelated to treatment with the test item. 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 behaviour, please refer to Attachment 1 - Tables 5 - 6 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):

At the end of treatment, test item-related liver weight findings were noted. Higher liver weight (statistically significant as absolute value and relative to body weight) was present in females at 800 mg/kg bw/day (absolute: + 27% and relative + 21% compared to controls) and correlated to minimal microscopic centrilobular hypertrophy. Following the recovery period, there was no statistically significant difference in the liver weight of the test item-treated females compared to the control animals. At the end of treatment, males at 800 mg/kg bw/day presented higher liver weight (statistically significant only as relative to body weight; +13%) without microscopic correlate. This was regarded as not test item-related, as this small difference was interpreted to represent a reflection of the slightly lower body weight noted in males the end of treatment (-8%) rather than a test item-related effect. At the end of treatment, females at 800 mg/kg bw/day had slightly higher statistically significant mean kidney weight (absolute +15%; relative to body weight +10%). This difference was considered not to be test item-related given the small magnitude of the change, and the absence of a macroscopic or microscopic correlate. Other statistically significant changes at the end of the treatment period included higher adrenal gland weight (relative to body weight +21%) and lower pituitary gland weight (absolute -11%) both in males at 800 mg/kg bw/day. These differences were considered not related to the test item given the small magnitude of the changes and the lack of macroscopic or microscopic correlate. Furthermore, these minor differences were statistically significant only as relative to body weight or absolute value.
Remaining organ weight differences observed at the end of the recovery period, including those that reached statistical significance (higher absolute and relative prostate gland weight in males at 800 mg/kg bw/day and lower absolute and relative thymus weight in females at 800 mg/kg bw/day), were considered incidental and unrelated to the administration of the test substance, as these differences occurred without macroscopic or microscopic correlate and were not present at the end of treatment in males or females of the main study. To sum up, changes in liver weight were interpreted to be treatment-related, but not adverse. Rather, they are regarded as an adaptive response.
For details on organ weights, please refer to Attachment 1 - Tables 11 - 12 under section "Attached background material".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, test item-related irregular surface was observed in the non-glandular stomach of 7/10 males and 9/10 females at 800 mg/kg bw/day. This finding was not observed after the end of the recovery period. Dark red focus in the glandular stomach was noted in one control male, one male at 300 mg/kg bw/day and five males at 800 mg/kg bw/day. This finding correlate to microscopic minimal acute mucosal hemorrhage in the control male and only three males at 800 mg/kg bw/day (the other males were without microscopic correlate). Given the acute nature of the microscopic correlate and the generally comparable incidence of this microscopic finding between the control and test item-treated animals, this was interpreted as unrelated to the test item. The remainder of the recorded macroscopic findings was 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 14 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 non-glandular stomach of males and females and the liver and thyroid gland of females.
Non-glandular stomach:
At the end of treatment, squamous cell hyperplasia with hyperkeratosis (diffuse to regionally extensive, up to moderate degree), was recorded in 8/10 males and 9/10 females at 800 mg/kg bw/day. Some males and females at 800 mg/kg bw/day presented a few other microscopic findings including minimal, focal mucosal erosion (often located at the limiting ridge), minimal to mild regionally extensive submucosal edema and minimal to mild submucosal mixed cell infiltrates. After the 28-day treatment-free period, minimal squamous cell hyperplasia with hyperkeratosis was noted only in 1/5 males. Edema in the non-glandular stomach of one female at 300 mg/kg bw/day was of low incidence and severity (1/8, minimal, regionally extensive) and although this finding did not appear in the concurrent controls, a similar low incidence can be found as background finding in rats of this age and strain, thus this finding was regarded to be unrelated to the test item. At 800 mg/kg bw/day, minimal acute mucosal hemorrhage (focal) was noted in 3/10 males, however, at this low incidence and severity a relationship to the test item was unlikely as this was present at generally comparable incidence in the control males. To sum up findings in the non-glandular stomach, the combination of these findings at the recorded incidences, severities and distribution are considered local adverse findings that resulted from the irritating properties of the test item rather than representing a systemic test item effect.
Liver:
At the end of treatment only, minimal centrilobular hypertrophy was present in 3/10 females at 800 mg/kg bw/day. This finding was not observed after the end of the recovery period and is therefore regarded as non-adverse.

Thyroid gland: At the end of treatment only, diffuse, bilateral minimal follicular cell hypertrophy was present in 3/10 females at 800 mg/kg bw/day. This finding was not present after the 28-day treatment-free period, which indicates recovery of the microscopic thyroid gland effects. The single incidence of minimal follicular cell hypertrophy noted in one female at 300 mg/kg bw/day was regarded as unrelated to the test item as this finding was within the normal range of background pathology encountered in rats of this age and strain. Minimal follicular cell hypertrophy was present at the end of the treatment period in 3/10 males at 800 mg/kg bw/day. This finding was regarded as not treatment-related, as this occurred at a comparable incidence in the control males (3/10 versus 3/10 in the controls). The
There were no other test item-related histologic changes.
For details on microscopic findings, please refer to Attachment 1 - Table 15 under section "Attached background material".

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm analysis:
No effects on sperm were observed in males up to 300 mg/kg bw/day. A decrease in progressive sperm cells was seen in males at 800 mg/kg bw/day at the end of the dosing period (0.72 x of control), which is regarded to be an adverse change.
For details on sperm analysis, please refer to Attachment 1 - Table 13 under section "Attached background material".

Details on results:

For details on the results, please refer to Attachment 1, under section "Attached background material".

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

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:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sperm measures

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:

800 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.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

other: progressive sperm cells

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

yes

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 90 days was well tolerated in rats at levels up to 300 mg/kg bw/day. Adverse test item-related morphologic alterations were present at 800 mg/kg bw/day in the non-glandular stomach of males and females. In addition, a decrease in progressive sperm cells was observed in males at 800 mg/kg bw/day. From the results presented in this report a No Observed Adverse Effect Level (NOAEL) systemic of 300 mg/kg bw/day for males and females and a NOAEL local of 300 mg/kg bw/day for males and females was established.

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 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.

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, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Studies with alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0)

Alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) 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 (Innospec, 2020). 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 29-38 days whereas females that delivered were treated for 50-67 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 42-43 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.

For females treated at 1000 mg/kg bw/day, slight to severe lethargy was noted during the entire study period. In the beginning of the study, the majority of the animals were affected and this was accompanied by abnormal, flat and hunched posture and uncoordinated movements. At the end of the study, these clinical signs were noted in individual animals and lateral recumbency, ventro-lateral recumbency, loss of righting reflex, decreased locomotor activity and abnormal gait were also noted in individual animals. Rales was noted for females treated at 1000 mg/kg bw/day for the majority of the animals during the entire study period, which was incidentally accompanied with slow breathing, laboured, deep and shallow respiration for individual animals. Furthermore, piloerection was also noted for females at 1000 mg/kg bw/day during the entire study period. Other clinical signs that were incidentally noted for these animals were salivation, chromodacryorrhoea, lean appearance and ptosis. Based on the incidence and severity of these clinical signs, these were considered adverse. At the end of the treatment period, an increased bile acids concentration was noted for females treated at 1000 mg/kg bw/day, in absence of histopathological correlation, which was considered not toxicologically relevant. No toxicologically significant changes were noted in any of the remaining parameters, i.e. mortality/moribundity, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, clinical laboratory investigations (hematology and clotting parameters), macroscopic examination, organ weights, and microscopic examination.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of 300 mg/kg bw/day for parental systemic toxicity was determined due to the severe and adverse clinical signs observed throughout the study period.

Alcohols, C9-11, branched and linear, ethoxylated (CAS 160901-09-7, EC No. 500-446-0) 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 (Innospec, 2022a). Groups of 10 animals/sex were administered doses of 100, 300 and 800 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 800 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.

Two mid-dose females were found dead after dosing on Day 8 and 13, respectively. No clinical signs were observed prior to their death. At necropsy, clear, watery fluid was noted in the thoracic cavity of both females. In addition, dark red discoloration of the left lung and dark red foci on the thymus were noted for one female and for the other, the lungs failed to collapse. There were no microscopic findings that could explain the cause of death. These premature deaths were considered to have resulted from an incident unrelated to the treatment with the test substance, given the lack of a dose response and in the absence of any other supporting findings.

During the dosing period, salivation was observed on several to many occasions shortly after dosing in 0/15, 7/10, 10/10 and 15/15 males in the control, low- mid- and high-dose group, respectively, and in 1/15, 6/10, 8/10 and 15/15 females in the control, low- mid- and high-dose group, respectively. The number of observations increased with the dose. No salivation was noted during the recovery period. The salivation was not considered toxicologically relevant and was seen as a physiological response rather than a sign of systemic toxicity. Abnormal breathing sounds were noted during the dosing period in 0/15, 1/10, 3/10 and 13/15 males in the control, low- mid- and high-dose group, respectively, and in 0/15, 0/10, 2/10 and 10/15 females in the control, low- mid- and high-dose group, respectively. The number of observations increased with the dose. This was also noted in 1/5 high-dose males and 2/15 high-dose females during the recovery period. Animals in a prostrate position was noted on several occasions on 4/15 high-dose males and 9/15 high-dose females only. Erected fur was observed once or a few times during the dosing period in 0/15, 1/10, 0/10 and 6/15 males in the control, low- mid- and high-dose group, respectively, and in 0/15, 0/10, 0/10 and 5/15 females in the control, low- mid- and high-dose group, respectively. Based on the limited occurrence and short duration, these observations were not considered to be toxicologically relevant. No toxicologically relevant clinical signs were noted during the weekly arena observations.

For males in the high-dose group, the body weight gain was slightly, but not significantly, lower (7%) than controls at the end of the dosing period (Day 91). Following the recovery period, the high-dose males showed a 4% lower mean body weight than for the controls (not statistically significant) on Day 123. The body weight changes in high-dose males was not considered to be toxicologically relevant. The body weight gain in females was similar to the control for all treatment groups.

The food consumption was slightly lower in high-dose males and females at several time points during the dosing period, compared with the control results (up to 8% lower than control, 2% lower over the whole dosing period; not statistically significant). This was not considered to be a treatment-related effect due to the small changes.

In high-dose males the red blood cell distribution width (RDWG) was statistically significantly decreased at the end of the dosing period (0.96 x) compared with the control. No difference between the control and high-dose value was recorded following the recovery period and the change is not considered treatment-related. At the end of the recovery period, monocyte (MONO) count and hemoglobin (HGB) concentration were statistically significantly decreased in high-dose males (0.70 and 0.95 x, respectively, compared with control). These are considered to be an incidental changes as they were observed only at the end of the recovery period.

Some minor changes in the clinical biochemistry parameters were noted at the end of the dosing period: Decreased triglyceride levels (0.76 x of control, not statistically significant) and increased potassium levels (1.09 x of control, statistically significant) were noted in high-dose males, compared with control. At the end of the recovery period, all parameters for the treatment groups were similar to control.

The thyroxine (T4) levels were increased in high-dose females compared with control (1.22 x, not statistically significant) at the end of treatment period. The thyroid stimulating hormone (TSH) levels were increased in mid- and high-dose males compared with control (1.40 and 1.27 x of control, respectively) and in females at all dose levels (2.17, 1.45 and 2.25 x, respectively) at the end of the dosing period, although without statistically significance. At the end of the recovery period, the mean TSH level was decreased in high-dose males (0.43 x; not statistically significant) but increased in females (1.44 x; not statistically significant), compared with control. In addition, Triiodothyronine (T3) concentration was increased in high-dose females (1.23 x; statistically significant) compared with control. All mean values at the end of the treatment and recovery period were well within the historical control data range. The recorded changes are therefore not considered to be treatment-related.

A significantly decreased percentage of progressive sperm was observed in males in the high-dose group following the treatment period: 28% versus 39% for control. The percentage was similar between the control and high-dose males in the recovery groups. This is considered to be a treatment-related effect.

In the neurobehavioural examination, the mean total movements of high-dose males was lower compared to control (0.67 x of control, statistically significant). This was considered the result of relatively high control values and as all values remained well within the range considered normal for rats of this age and strain. This difference was considered to be unrelated to treatment with the test substance.

In the main group, a significant increase in absolute liver weight was noted in high-dose females (26%), compared with the controls. The increase in relative liver weight was statistically significant in high-dose males (12%) and in high-dose females (21%), compared with control. 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 statistically significant increase in absolute kidney weight was noted in high-dose females (15%) of the main group, compared with the control. The increase in absolute kidney weight was also statistically significant in high-dose females (10%). Following the recovery period there was no major difference in kidney weight in either sex.

Macroscopic examination showed an irregular surface of the non-glandular stomach 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 9/10 females in the control, low- mid- and high-dose group, respectively, following the dosing period. In the glandular stomach, dark red foci in the mucosa was noted in 1/10, 0/10, 1/10 and 5/10 males in the control, low- mid- and high-dose group, respectively, following the dosing period. Microscopic lesions were also noted in high-dose animals. At the end of the recovery period, 1/5 high-dose males had dark red foci in the glandular mucosa.

Treatment-related microscopic findings were noted in the thyroid gland, liver and stomach of males and females, and in the kidney of males. Centrilobular hepatocellular hypertrophy was noted in 0/10, 0/10, 0/10 and 3/10 females in the control, low- mid- and high-dose group, respectively, following the dosing period. The effect is likely correlated to the higher liver weights. Centrilobular hypertrophy was not observed in males or females after the recovery period, indicating a reversible adaptive response.

Several findings were noted in the non-glandular stomach (forestomach) following the dosing period. Squamous cell hyperplasia (minimal to mild) 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, 0/10, 0/10 and 9/10 females in the control, low- mid- and high-dose group, respectively. Minimal erosion was noted in 2/10 males and 1/10 females in the high-dose group only. Submucosal edema (minimal to mild) was observed in 0/10, 0/10, 0/10 and 2/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 1/10 and 2/10 females in the control, low- mid- and high-dose group, respectively. In the non-glandular stomach mixed-cell submucosal infiltration (minimal to mild) was observed in 1/10, 0/10, 0/10 and 7/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 4/10 females in the control, low- mid- and high-dose group, respectively. Hyperkeratosis (minimal to moderate) was noted in 8/10 males and 9/10 females in the high-dose group only. In the glandular stomach, acute mucosal haemorrhage was noted in 1/10, 0/10, 1/10 and 3/10 males in the control, low- mid- and high-dose group, respectively, following the dosing period. The few occurrences in the non-glandular and glandular stomach of low-, and mid-dose animals were not considered to be clear indications of a treatment-related effect. Following the recovery period, 0/5 and 1/5 males in the control and high-dose group, respectively, showed squamous cell hyperplasia of the non-glandular stomach. Hyperkeratosis (minimal) of the non-glandular stomach was noted in 1/5 males in the high-dose group only. In the glandular stomach, acute mucosal haemorrhage was noted in 0/5 and 1/5 males in the control and high-dose group, respectively, and 1/5 and 0/5 females in the control and high-dose group, respectively. Due to the correlated macroscopic and microscopic findings in the forestomach, these are considered toxicologically relevant in the high-dose group.

In males, hyaline droplet accumulation was noted in the kidneys following the dosing period. This was observed in 5/10, 0/1 and 7/10 males in the control, low- and high-dose group, respectively. The kidneys of one of the low-dose males and none of the mid-dose males were examined. Following the recovery period 1/5 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 follicular cell hypertrophy was noted following the dosing period in 3/10 and 3/10 males in the control and high-dose group, respectively, and in 0/10, 0/10, 1/8 and 3/10 females in the control, low- mid- and high-dose group, respectively. The thyroid glands of the male low- and mid-dose groups were not examined. Following the recovery period, no animals had follicular cell hypertrophy. This indicates a recovery of the microscopic thyroid gland effects.

The results of the opthalmoscopic examination, urinalysis and estrous cycle determination 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 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, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) 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, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) 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.