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Administrative data

Description of key information

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

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

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

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

 

Conclusion based on data obtained with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Nov 2019 - 03 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 (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. 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
- Age at study initiation: Males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: Males 272-340 g, females 198-249 g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 20-59
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 5 FEB 2020 to 30 MAR 2020
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human
exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- 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
- Batch: MKCH1635 + MKCG3257, MKCK6411
- 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 (UPLC-MS ).
- Concentration analysis was conducted. Results were considered acceptable if mean sample
concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of
variation (CV) of concentrations was less than or equal to10%.
- Stability analyses were performed previously in conjunction with the method development and
validation study (Test Facility Study No. 20218713).
Duration of treatment / exposure:
Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days.
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 Finding Study (Test Facility Reference No. 20218714) with oral gavage administration of Alcohols, C12-14, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

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

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

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

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

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity.
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, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid,
gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea, urinary bladder, uterus, vagina.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Datasets with at least 3 groups 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):
No toxicologically relevant clinical signs were noted during daily detailed clinical
observations and no findings were noted during the weekly arena observations in this study.
In females at 1000 mg/kg bw/day, piloerection was incidentally noted, and uncoordinated
movements and rales were noted once each. Based on the incidence observed this was
considered not to be toxicologically relevant.
Salivation was seen after dosing from 300 mg/kg bw/day upwards in a dose-related trend.
This was considered not to be 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.
Incidental findings that were noted included alopecia, piloerection, abdominal swelling,
abdominal nodule and a broken tail apex. These findings occurred within the range of
background findings to be expected for rats of this age and strain which are housed and
treated under the conditions in this study. At the incidence observed, these were considered
not to be signs of toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes in body weight and body weight gain were observed up
to 1000 mg/kg bw/day.
A reduced body weight gain was noted for males treated at 1000 mg/kg bw/day during the
first two weeks of treatment. At the end of the study period absolute body weight was 5% lower compared to controls. Based on the magnitude of the change this was considered not to
be toxicologically relevant.
Body weights and body weight gain of males up to 300 mg/kg bw/day and females up to
1000 mg/kg bw/day remained in the same range as controls over the treatment period.
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 recorded.
A slightly lower absolute food consumption was noted during the last week of lactation for
females treated at 1000 mg/kg bw/day (-14% compared to control). Relative food
consumption was reduced from Day 4 of lactation onwards for these animals (up to -12%
compared to control). Based on the magnitude of the change, this was considered not to be
toxicologically relevant.
Any other statistically significant changes in food consumption before or after correction for
body weight were considered to be unrelated to treatment since no trend was apparent
regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematology parameters of treated rats were considered not to have been affected by treatment
with the test item.
Any statistically significant changes in hematology parameters were considered to be
unrelated to treatment as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment
with the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
An increased alkaline phosphatase (ALP) activity (1.43x of control) and albumin
concentration (1.06x of control) were noted for males treated at 1000 mg//kg/day.
Additionally, a decreased concentration of total bilirubin (up to 0.82x of control) was noted
for males treated at 100, 300 and 1000 mg/kg bw/day, reaching statistical significance for
males at 1000 mg/kg bw/day. For females, an increased concentration of calcium was noted at
300 and 1000 mg/kg bw/day (1.06x of control for both dose levels). Based on the magnitude
of the change, slightly low control values, absence of a dose-related trend and/or as mean
values remained within the historical control ranges, these changes were considered not to be
toxicologically relevant.
Any other statistically significant changes in clinical chemistry parameters were considered to
be unrelated to treatment as these occurred in the absence of a dose-related trend.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of T4 in F0-males at 1000 mg/kg bw/day were decreased (0.75x of control).
Mean values were within the range of historical control values.
No treatment-related effect was noted on serum levels of T4 in F0-females and serum levels
of TSH in F0-males and females.
Under the conditions of this screening study no associated adverse effect with the decrease in T4 levels in F0 males was observed and so these findings were not taken into account when determining the parental NOAEL.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined
animals up to 1000 mg/kg bw/day. Grip strength was similar between control and treated
animals.
A decreased number of total movements and ambulations (not statistically significant) was
noted for females treated at 1000 mg/kg bw/day. As mean values were within the historical
control range, this was considered not to be toxicologically relevant.
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 weights (absolute and/or relative to body weights) were noted in the
male liver and kidney at 300 and 1000 mg/kg bw/day and female adrenal gland at 1000 mg/kg
bw/day as shown in the Table 1.
A higher liver weight (absolute and relative to body weight) was recorded for males at 300
and 1000 mg/kg bw/day. The microscopic correlate at 1000 mg/kg bw/day was centrilobular
hepatocellular hypertrophy.
Higher kidney weights were recorded for males at 300 mg/kg bw/day (absolute) and at
1000 mg/kg bw/day (absolute and relative to body weight). There was no microscopic
correlate for this weight change.
Higher adrenal gland weights were recorded for females at 1000 mg/kg bw/day (absolute and
relative to body weights). The microscopic correlate for this weight change was cortical
hypertrophy, zona fasciculata.
There were no other test item-related organ weight changes. Some organ weight differences
were statistically significant when compared to the control group; a lower absolute prostate
gland weight and higher testes and epididymides weight relative to body weight at 1000
mg/kg bw/day. This was considered to be the result of a test item-related lower final body
weight in males at 1000 mg/kg bw/day. As changes were slight this was considered not
adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
An accentuated lobular pattern in the liver was recorded for a single male at 100 mg/kg
bw/day, two males at 300 mg/kg bw/day and three males at 1000 mg/kg bw/day. At 1000
mg/kg bw/day the microscopic correlate for this finding was centrilobular, hepatocellular
hypertrophy. This finding was interpreted to be non-adverse at the recorded minimal severity
and in absence of any additional inflammatory or degenerative alterations.
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 after treatment with Alcohols, C12-14, ethoxylated
(2 EO) were noted at 1000 mg/kg bw/day in the stomach of both sexes, liver and jejunum of
males and adrenal gland of females.
Squamous cell hyperplasia at minimal degree was noted in the forestomach of a single male
and two females at 1000 mg/kg bw/day, shown in Table 2.
Centrilobular hepatocellular hypertrophy of the liver at minimal degree was noted in 4/5
males at 1000 mg/kg bw/day. This was the microscopic correlate to the higher liver weights
and the accentuated lobular pattern recorded at this dose level.Vacuolation of the jejunum was recorded in three males at 1000 mg/kg bw/day. These round,clear vacuoles were present in the lamina propria of the villi of the jejunum, shown in Table 3.
Cortical hypertrophy of the zona fasciculata of the adrenal gland was recorded in 4/5 females
at 1000 mg/kg bw/day at minimal degree, shown in Table 4.
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.


Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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

Table 1
Mean Percent Liver and Kidney Weight Differences from Control Groups

 

Dose level (mg/kg/day):

100

300

1000

LIVER (Males)

 

 

 

               Absolute

11

24**

37**

               Relative to body weight

7

18**

40**

 

 

 

 

KIDNEY (Males)

 

 

 

               Absolute

9

13*

11*

               Relative to body weight

5

8

13**

 

 

 

 

ADRENAL GLANDS (Females)

 

 

 

               Absolute

7

-3

27*

               Relative to body weight

4

0

27*

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

Table 2
Summary Test Item-Related Microscopic Findings – Both sexes

 

Males

Females

Dose level (mg/kg/day):

0

100

300

1000

0

100

300

1000

 

 

 

 

 

 

 

 

 

STOMACH a

5

5

5

5

5

5

5

5

    Hyperplasia, squamous cell,

                                 forestomach

 

 

 

 

 

 

 

 

       Minimal

-

-

-

1

-

-

-

2

 

 

 

 

 

 

 

 

 

a  =  Number of tissues examined from each group.

Table 3
Summary Test Item-Related Microscopic Findings – Males

 

Males

Dose level (mg/kg/day):

0

100

300

1000

 

 

 

 

 

LIVER a

5

6

6

5

     Hypertrophy, hepatocellular,

                                centrilobular

 

 

 

 

       Minimal

-

-

-

4

 

 

 

 

 

JEJUNUM a

5

5

5

5

    Vacuolation

 

 

 

 

       Minimal

-

-

-

3

 

 

 

 

 

a  =  Number of tissues examined from each group.

Table 4
Summary Test Item-Related Microscopic Findings – Females

 

Females

Dose level (mg/kg/day):

0

100

300

1000

 

 

 

 

 

ADRENAL GLAND a

5

5

5

5

     Hypertrophy cortical,

                            zona fasciculata 

 

 

 

 

       Minimal

-

-

-

4

 

 

 

 

 

a  =  Number of tissues examined from each group.

 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Jun - 09 Dec 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
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: Carles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 150 - 201 g (males) and 107 - 135 g (females)
- Fasting period before study: no
- 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) and 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: 13 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 22
- Humidity (%): 45 - 69
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 22 Jun - 19 Oct 2021
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared at least weekly and stored at 4 °C. Formulations (w/w) were homogenized to visually acceptable levels by stirring continuously during dosing. An adjustment was made for the specific gravity of the test item and the vehicle. No correction was made for purity / composition of the test item. The dosing volume was 5 mL/kg bw, calculated and adjusted based on the most recent body weight measurement.

VEHICLE
- Justification for use and choice of vehicle: test item stable in vehicle, as demonstrated by stability analysis; trial preparations were performed and showed suitability of the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL for the low-, mid- and high-dose group, respectively.
Analytical verification of doses or concentrations:
yes
Remarks:
in Weeks 1, 6 and 12
Details on analytical verification of doses or concentrations:
Accuracy: The concentrations analyzed in the Group 2 - 4 formulations were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85 - 115% of target concentration). In the chromatograms of the Group 1 formulation prepared for use in Week 1, a small response at the retention time of the test item was observed. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.
In the chromatograms of the Group 1 formulation prepared for use in Week 6, no test item was detected. In one of the chromatograms of the Group 1 formulation prepared for use in Week 12, a small response at the retention time of the test item was observed. It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. The maximum contribution to the Group 2 samples was 0.019%, taking the dilution factor into account. As this was only a very small response, this result will not have an impact on the study.
Homogeneity: The Groups 2 and 4 formulations prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Main study: 13 weeks
Recovery: 28 days
Frequency of treatment:
once daily, 7 days / week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
Main study: 10 (Group 1 - 4)
Recovery groups: 5 (Group 1 and 4)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily; from Day 1 at 0 - 1 h postdose; twice daily checked for mortality and moribundity

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

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

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

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

WATER CONSUMPTION: Yes (qualitatively)
- Time schedule for examinations: on 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 3
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) and 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), and in Week 17 of the recovery period
- Dose groups that were examined: first 5 animals per sex per group; all recovery animals
- 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.
Other examinations:
-Estrous stage determination: At the end of treatment or recovery on the day of sacrifice, a vaginal smear will be taken to determine the stage of estrous.
- Sperm analysis: from all surviving males; evaluated for sperm morphology and sperm numbers
Statistics:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test, ANCOVA, Fisher's exact test and Steel-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations in the dosing and recovery period.
Salivation occurred 3/10 males and 1/10 female in the 100 mg/kg bw/day group, in 9/10 males and 8/10 females in the 300 mg/kg bw/day group, and in all males and females of the 1000 mg/kg bw/day group. Salivation 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 to dosing rather than a sign of systemic toxicity.
Abnormal breathing sounds were noted in 1/10 males at 100 mg/kg bw/day on a single day, and 10/15 males and 8/15 females at 1000 mg/kg bw/day on up to nine consecutive days on one or more occasions. Deep breathing was observed in 2/15 males and 1/15 female at 1000 mg/kg bw/day on one to two days. As these clinical signs were observed for only relatively short periods without any relation to the duration of dosing and animals recovered, this was considered not to be toxicologically relevant.
Hunched posture was observed incidentally in 1 male and female at 1000 mg/kg bw/day. Erected fur was incidentally observed in 1/10 males at 100 and 300 mg/kg bw/day each, and 4/15 males and 1/15 female at 1000 mg/kg bw/day. Based on the low incidence, short duration of the clinical signs and/or since animals recovered, these were considered to be not toxicologically relevant.
One female at 1000 mg/kg bw/day (No. 94) was observed with red discharge from the mouth, muzzle and nostrils on Day 84 (together with hunched posture). As this occurred on a single day in only one animal, this is considered to be unrelated to the dosing with the test item.
Other findings (e.g., fur loss, scabs and skin lesions) noted during the dosing and/or recovery period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
For details on clinical observations, please refer to Attachment 1 - Table 1 under the section "attached background material".
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one premature death in the study, which was considered unrelated to the test item: animal No. 27 (male dosed at 300 mg/kg bw/day) was found dead on Day 75 of the study. This animal was seen with slight salivation at 0 – 1-hour post-dose on Days 14 - 16 and Day 57, but no other clinical signs were observed. At necropsy, enlargement and dark red discoloration of the papillary process of the liver were noted, which correlated microscopically with diffuse coagulative necrosis isolated to the papillary process (with haemorrhage and fibrin) and is consistent with a spontaneous liver lobe torsion. Dark red foci were noted in the thymus, but this was without a microscopic correlate. In addition, there were no microscopic findings of note that could explain the cause of death of this animal.
This premature death was considered unrelated to the treatment with the test item given the lack of a dose response and the absence of adverse systemic test item-related effects in the animals dosed at 1000 mg/kg bw/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of males and females at 100 and 300 mg/kg bw/day remained in the same range as controls over the study period.
Reduced body weight gain was observed in males at 1000 mg/kg bw/day over the entire dosing period compared with control, reaching statistical significance from Day 57 onwards, with the body weight being 12 % lower on Day 91 in animals dosed at 1000 mg/kg bw/day compared to control. Partial recovery was observed during the dosing-free period (Days 92 - 119), with statistically significantly lower body weight on Days 92 and 99 only, compared with control. At the end of the recovery period, mean body weight in males at 1000 mg/kg bw/day was 5% lower than in control.
A slight increase in body weight gain was observed in females at 1000 mg/kg bw/day over the entire dosing period compared with control, with a statistically significant body weight gain over Days 8 - 15 and 78 - 85 resulting in statistically significant higher body weight on Days 15 - 29 (7% increase on Days 15, 22 and 29) and on Day 85 (7% increased). At the end of the recovery period, mean body weight in females at 1000 mg/kg bw/day was 3% higher than in control. This slightly higher body weight gain in comparison with the control group was at a magnitude of change considered not to be toxicologically relevant. Based on the relatively slight severity, the opposite effect in males and females, and the full recovery, this body weight (gain) effect was regarded to be not adverse.
For details on body weight and body weight changes, please refer to Attachment 1 - Figure 1 and Tables 2 - 3 under the section "attached background material".
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption were recorded in males up to 1000 mg/kg bw/day and females at 100 and 300 mg/kg bw/day. The apparent increase in food consumption of males dosed with the test item between Days 43 - 50 was caused by a decreased food consumption in control animals. Females at 1000 mg/kg bw/day had an increased food consumption from Day 8 onwards (without reaching statistical significance), with an overall increased food consumption of 9.3% compared to control animals over the whole duration of the dosing period (Day 1 - 91). During the recovery period, food consumption of females dosed at 1000 mg/kg bw/day was still slightly higher (6.7% compared to controls between Days 92 - 119). This slightly higher food consumption in comparison with the control group was at the a magnitude of change considered not to be toxicologically relevant.
For details on food consumption, please refer to Attachment 1 - Figure 2 and Table 4 under the section "attached background material".
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The results described below are statistically significant unless specified:
Main groups: Hematological parameters of males up to 300 mg/kg bw/day and females at 100 mg/kg bw/day were considered not to have been affected by treatment with the test item.
Neutrophil (NEUT) and monocyte (MONO) counts were increased in males at 1000 mg/kg bw/day (1.44 and 1.64 x of control, respectively), causing a slight increase in white blood cell count (WBC; 1.20 x, not statistically significant).
Red blood cell (RBC) count was increased in males at 1000 mg/kg bw/day (1.05 x) and females at 300 and 1000 mg/kg bw/day (1.10 x for both). In males this increase in RBC count was caused by a slightly higher reticulocyte (RETIC) count (1.13 x, not statistically significant). Furthermore, in females at 300 and 1000 mg/kg bw/day an increase in hemoglobin (HGB; 1.07 and 1.08 x, respectively) and hematocrit (HCT; 1.09 and 1.10 x, respectively) was observed.
Platelet count (PLT) was increased in females at 300 and 1000 mg/kg bw/day (1.24 and 1.18 x, respectively).
Recovery groups: At the end of the recovery period, all hematological parameters in males and most parameters in females had recovered. Only HGB remained increased in females at 1000 mg/kg bw/day (1.04 x).
White blood cell (WBC) was increased in females at 300 mg/kg bw/day (1.51 x of control), caused by an increase in lymphocyte (LYMPH) and MONO count (1.57 and 1.37 x, respectively; not statistically significant for MONO). In absence of a dose response, this is regarded to be not test item-related.
Platelet-clumps were seen in two males at 300 mg/kg bw/day and two females at 1000 mg/kg bw/day, at the end of the dosing period only. At this low occurrence, and in absence of a clear dose response, this was considered to be not test item-related.
Based on the reversibility, type of change (i.e. higher HGB), the low severity and/or in absence of a histopathological correlation, the listed changes in hematology parameters were considered to be non-adverse.
Remaining differences in hematology parameters, including changes at the end of the recovery period, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
For details on haematology, please refer to Attachment 1 - Table 7 under the section "attached background material".
Coagulation: No effects on coagulation parameters were observed in males up to 1000 mg/kg bw/day and females up to 300 mg/kg bw/day.
Activated partial thromboplastin time (APTT) was shorter in females at 1000 mg/kg bw/day (0.88 x of control). At the end of the Recovery Period, APTT values were comparable to controls. In absence of a histopathological correlation, this finding was considered to be non-adverse.
For details on coagulation, please refer to Attachment 1 - Table 8 under section "attached background material".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The results described below are statistically significant unless specified:
Main groups: Changes in clinical chemistry parameters comprised an increase in alanine aminotransferase (ALT) activity in males at 100, 300 and 1000 mg/kg bw/day (1.24, 1.16 and 1.35 x of control, respectively; not statistically significant at 300 mg/kg bw/day), increased aspartate aminotransferase (AST) in males at 100, 300 and 1000 mg/kg bw/day (1.29, 1.23 and 1.48 x, respectively; not statistically significant at 100 and 300 mg/kg bw/day) and alkaline phosphatase (ALP) activity in males and females at 1000 mg/kg bw/day (1.30 and 1.32 x, respectively; not statistically significant in females).
Urea (UREA) was increased in males at 100, 300 and 1000 mg/kg bw/day (1.36, 1.52 and 1.61 x, respectively) and females at 100, 300 and 1000 mg/kg bw/day (1.21, 1.32 and 1.46 x, respectively).
In males at 100, 300 and 1000 mg/kg bw/day, a decreased level of cholesterol (CHOL; 0.85, 0.77 and 0.73 x, respectively; not statistically significant at 100 mg/kg bw/day), HDL cholesterol (HDL; 0.86, 0.78 and 0.68 x, respectively) and triglycerides (TRIG; 0.61, 0.56 and 0.48 x, respectively) was observed.
Potassium (K) and inorganic phosphate (PHOS) levels were increased in males at 1000 mg/kg bw/day (1.11 and 1.15 x, respectively), and chloride (CL) levels were decreased in males and females at 1000 mg/kg bw/day (0.98 and 0.97 x, respectively).
Recovery groups: In males, creatinine (23.7 µmol/L) and inorganic phosphate (1.9 mmol/L) were increased, while in females, ALT (37.2 U/L) and AST (77.0 U/L) were higher.
Remaining differences in clinical chemistry parameters, regardless of statistical significance, were considered not to be test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, occurred during the recovery period only, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
These parameters were considered non-adverse based on the absence of any histopathological correlation, opposite effect observed at the end of the recovery period, and/or (partial) recovery of the parameters after the recovery period.
For details on clinical biochemistry, please refer to Attachment 1 - Table 9 under the section "attached background material".
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Main groups: At 1000 mg/kg bw/day, thyroxine (T4) was decreased in males (0.80 x), triiodothyronine (T3) concentration was decreased in females (0.77 x) and thyroid stimulating hormone (TSH) concentration was increased in males and females (1.85 and 1.65 x, respectively; not statistically significant).
Recovery groups: After the recovery period, TSH levels were decreased in males at 1000 mg/kg bw/day (0.26 x) while this was increased at the end of the dosing period (1.85 x). All other values returned to normal values for both males and females. These parameters were considered non-adverse based on the absence of any histopathological correlation, opposite effect observed at the end of the recovery period, and/or the (partial) recovery of the parameters after the recovery period.
For details on thyroid hormone levels, please refer to Attachment 1 - Table 9 under the section "attached background material".
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
No effects on urine volume were noted in males and females up to 300 mg/kg bw/day. In both males and females, the mean urine volume was increased at 1000 mg/kg bw/day (2.24 x and 2.52 x of control, respectively) at the end of the dosing period. At the end of the recovery period, urine volume was comparable between animals dosed with the test item and controls. The effect was thus considered non-adverse.
No effect was observed on the pH of the urine.
For details on urinalysis, please refer to Attachment 1 - Table 10 under the section "attached background material".
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Main / Recovery groups: Hearing ability, pupillary reflex and static righting reflex were normal in all examined males and females. Grip strength was similar between control and females at 1000 mg/kg bw/day.
Decreased grip strength in the fore and hind legs was observed in males at 1000 mg/kg bw/day (0.78 and 0.71 x, respectively) at the end of the dosing period. At the end of the recovery period, grip strength had recovered and was similar between males at 1000 mg/kg bw/day and control groups.
For the main groups, a decreased number of total movements and ambulations was observed for females at 100 and 1000 mg/kg bw/day (0.68 and 0.53 x, respectively for total movements; and 0.63 and 0.54 x, respectively for ambulations), without a clear dose response. At the end of the recovery period, a decreased number of total movements and ambulations was still present in females at 1000 mg/kg bw/day (0.72 x) compared to controls. The number of total movements and ambulations was increased in males at 100 mg/kg bw/day, without a clear dose response. Furthermore, at the end of recovery period, the number of total movements and ambulations was slightly increased for males at 1000 mg/kg bw/day compared to controls. Based on the absence of a dose-response and the opposite effect observed in males and females, this was regarded to be not test item-related.
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 behaviour, please refer to Attachment 1 - Tables 5 - 6 under the section "attached background material".
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher kidney weight was present in females at 300 and 1000 mg/kg bw/day, and higher liver weight was present in males and females both starting at 300 mg/kg bw/day.
Liver:
Higher liver weight (statistically significant as absolute and relative to body weight) was present in males at 1000 mg/kg bw/day (11.339 g) and females at 300 and 1000 mg/kg bw/day (6.8308 g and 8.7474 g, respectively). In males, higher liver weight (statistically significant as relative to body weight only) was present at 100 mg/kg bw/day (2.56676 %bw), however this small difference was in line with the decrease in body weight of this dose group.
Following the recovery period, there was no significant difference in the liver weight of test item-treated males and females compared to the control animals, suggesting full recovery and a non-adverse effect.
Kidney:
Higher kidney weight (statistically significant as absolute and relative to body weight) was noted in females at 300 and 1000 mg/kg bw/day (1.5649 g and 1.6515 g, respectively). In males, apparent higher kidney weight (statistically significant as relative to body weight only) was observed at 300 mg/kg bw/day (main group: 0.649 %bw)and 1000 mg/kg bw/day (main group: 0.68917 %bw; recovery group: 0.64825 %bw). However, these differences were interpreted to be due to the lower terminal body weight in these treatment groups.
Following the recovery period, there was no significant difference in the kidney weight of test item-treated females compared to the control females, suggesting full recovery and therefore a non-adverse effect.
Miscellaneous:
Differences in terminal body weights of animals dosed at 1000 mg/kg bw/day (lower in males and higher in females) resulted in apparent organ weight differences of statistical significance and are described below.
At the end of dosing in males, apparent higher brain, adrenal gland, heart and testis weights were observed at 1000 mg/kg bw/day (relative to body weight only), but these were a result of the lower terminal body weight. At the end of recovery in males, similar to the other organs at 1000 mg/kg bw/day, the apparent higher brain weight (absolute and relative to body weight) was interpreted to be due to the difference in terminal body weight.
At the end of dosing in females, apparent higher adrenal gland and thyroid gland weights observed at 1000 mg/kg bw/day (absolute weight only) were a reflection of the higher terminal body weight; in addition, there was no significant difference when expressed relative to body weight.
For details on organ weights, please refer to Attachment 1 - Tables 12 - 13 under the section "attached background material".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A test item-related irregular surface was observed in the non-glandular stomach of 2/10 males at 1000 mg/kg bw/day at the end of the dosing period. This finding was not observed after the end of the recovery period.
Miscellaneous:
Prominent lobular architecture of the liver was recorded in 3/10 males at 100 and 300 mg/kg bw/day each, 2/10 males and females at 1000 mg/kg bw/day, and 1/5 recovery male at 1000 mg/kg bw/day. The relationship of this finding to the test item was unclear given the lack of a dose relationship noted in males and the low incidence present in females. Small seminal vesicles were observed in one male at 1000 mg/kg bw/day. This single finding was regarded to be related to the lower final body weight of this dose group (15%). Tan gelatinous content was noted in the kidney of one male at 1000 mg/kg bw/day. This finding correlated to renal nephroblastoma. Pale/pale tan discoloration of the liver was noted in 1 male in the control, 100 and 300 mg/kg bw/day groups each, and in 2/15 males at 1000 mg/kg bw/day. This lesion occurred without microscopic correlate. Given the generally comparable incidence of this macroscopic finding between the control and test item-treated animals, this was interpreted as unrelated to the test item.
Dark red foci in the glandular stomach were noted in 1/10 males at 100 mg/kg bw/day and 5/15 males at 1000 mg/kg bw/day. Although this finding was not present in control animals of this study, similar incidence and severity can be encountered in rats of this age and strain; thus, the relationship to the test item was interpreted as not likely. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
For details on gross pathological 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 liver, thyroid gland, stomach and jejunum of males and females, and kidney of males.
Liver:
Centrilobular hepatocellular hypertrophy (minimal) was present in 5/10 males and 5/10 females at 1000 mg/kg bw/day, at the end of the dosing period. Following the recovery period, this finding was not present in males or females, suggesting full recovery. These changes were considered non-adverse based on the low severity, reversibility, only minor alterations in liver-related blood chemistry, and the absence of associated degenerative, inflammatory or necrotic changes.
Thyroid gland:
An increased incidence and/or severity of diffuse, bilateral follicular cell hypertrophy (up to mild degree) was present in 7/10 males and 6/10 females at 1000 mg/kg bw/day, at the end of the dosing period. Following the recovery period, follicular cell hypertrophy (minimal) was present in 2/5 males and 1/5 females at 1000 mg/kg bw/day, but at this low incidence and severity a relationship to the test item was unlikely as this finding can be encountered in control rats of this age and strain, suggesting full recovery in both sexes. The low incidences of follicular cell hypertrophy (minimal) noted in animals of the remaining dose groups were interpreted as unrelated to the test item given the generally comparable incidence of this microscopic finding between the control and test item-treated animals. This finding was considered non-adverse based on the low severity, reversibility and the absence of associated degenerative, inflammatory or necrotic changes.
Gastro-intestinal tract: At the end of the dosing period, squamous cell hyperplasia with hyperkeratosis (diffuse to regionally extensive, up to mild degree), was recorded in 1/10 males and 1/10 females at 300 mg/kg bw/day, and in 10/10 males and 7/10 females at 1000 mg/kg bw/day. A few males and females at 300 and 1000 mg/kg bw/day presented a few other microscopic findings including, focal mucosal erosion (minimal, often located at the limiting ridge), submucosal edema (up to mild degree) and submucosal mixed cell infiltrates (minimal). Following the recovery period, squamous cell hyperplasia (focal, minimal) was still present in 1/5 females at 1000 mg/kg bw/day suggesting nearly complete recovery. These findings were not present in males, suggesting full recovery. The combination of these findings at the recorded incidences, severities and distribution are considered non-adverse and likely resulted from the irritating properties of the test item.
Dark red foci in the glandular stomach were noted in 1/10 male at 100 mg/kg bw/day and 5/10 males at 1000 mg/kg bw/day. This finding correlated to microscopic minimal acute mucosal hemorrhage in only three males at 1000 mg/kg bw/day (the others were without microscopic correlate). Although this finding was not present in control animals of this study, similar incidence and severity can be encountered in rats of this age and strain, thus, the relationship to the test item was interpreted as not likely.
In the jejunum, multifocal villous vacuolation (up to mild degree) was present in 10/10 males and 3/10 females at 1000 mg/kg bw/day, at the end of the dosing period. This finding was characterized by the presence of variable sized clear vacuoles in the lamina propria of the villi, that occasionally contained a minimal amount of lacy flocculent eosinophilic to amphophilic material. Following the recovery period, villous vacuolation (up to moderate degree in males and up to mild degree in females) was still present in 4/5 males and 5/5 females, suggesting no recovery in both sexes. Despite the absence of reversibility, and slightly increased incidence and/or severity presented in the recovery animals, this finding was regarded as non-adverse given the lack of associated degenerative, inflammatory, necrotic or clinical pathology changes.
Kidney:
In the kidney, at the end of the dosing period, increased incidence and severity of hyaline droplet accumulation (up to moderate degree) was recorded in 9/10 males at 1000 mg/kg bw/day. Following the recovery period, a background incidence and severity of hyaline droplet accumulation was recorded in 3/5 males (minimal) at 1000 mg/kg bw/day and 1/5 males (minimal) of the control group, which was interpreted as full recovery. Given the reversibility and absence of concurrent degenerative, inflammatory or necrotic changes, the alterations in the kidney of males and females were considered non-adverse. In general this finding is regarded as specific to male rats and not relevant to humans.
Microscopic findings of note:
In the pancreas of males and females, there was minor variation in the incidence of minimal depletion of acinar secretory content across groups, however this was without a clear dose relationship in males, and it occurred at a generally comparable incidence between the control and test item-treated females; thus, these differences were interpreted as not test item-related.
The remainder of the recorded microscopic findings 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 microscopic observations, please refer to Attachment 1 - Table 15 under the section "attached background material."
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A nephroblastoma was recorded in Animal No. 37 (male dosed at 1000 mg/kg bw/day). This finding correlated with the macroscopic observation of a tan gelatinous content in the kidney. The tumor was located within the renal pelvis and most likely originated at this region. This was characterized by the presence of highly basophilic, densely packed, blast cells. Nephroblastoma is uncommon among spontaneous tumors in rats, and it has been induced in the rat only with genotoxic chemicals, usually by transplacental, prenatal exposure (Frazier et al., 2012, Ito et al., 2014). Although the location of this particular tumor is unusual, this observation was interpreted to be a spontaneous change and not related to the test item given the single incidence in this study.
Other effects:
no effects observed
Description (incidence and severity):
Estrous stage determination: no effects observed
Sperm Analysis: no effects observed
For details on sperm analysis, please refer to Attachment 1 - Table 11 under section"attached background material"
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 adverse effects observed up to and including the highest dose level
Key result
Critical effects observed:
no
Conclusions:
The present study was conducted under GLP and according to OECD test guideline 408 (2018). Under the conditions of the study, administration of the test substance once daily by oral gavage for 90 days was well tolerated in rats at levels up to the highest dose level of 1000 mg/kg bw/day. All test item-related effects were considered non-adverse. From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day for males and females was established.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable (Klimisch score 1) studies performed with member substances of the Alcohol Ethoxylates (AE) category including data on the target substance. The selected studies are sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.6, of the REACH Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data on repeated dose toxicity are available for alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Studies with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3)

Subacute data

Alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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 (Sasol, 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 days whereas females that delivered were treated for 50-54 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 42-51 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.

Test item-related macroscopic, microscopic and/or organ weight changes were observed for animals at 300 and 1000 mg/kg bw/day. Squamous cell hyperplasia at a minimal degree was noted in the forestomach of a single male and two females at 1000 mg/kg bw/day, which is suggestive of (low) irritating properties of the test item. This change is a local test item-related finding and regarded to be non-adverse at this low severity and in absence of any additional inflammatory or degenerative alterations. Other microscopic findings were centrilobular hepatocellular hypertrophy in the liver and vacuoles in the jejunum of males at 1000 mg/kg bw/day and cortical hypertrophy, zona fasciculata of the adrenal glands (with correlating higher weights) of females at 1000 mg/kg bw/day. These findings were interpreted to be non-adverse at the recorded minimal severity and in absence of any additional inflammatory or degenerative alterations. Additionally, higher kidney weights were recorded in males at 300 and 1000 mg/kg bw/day, which were considered to be non-adverse in absence of any microscopic correlate. Test item-related non-adverse decreased serum levels of T4 were noted for F0-males at 1000 mg/kg bw/day. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption and hematology and coagulation parameters).

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

Subchronic data

Alcohols, C12-14, ethoxylated (CAS 68439-50-9, 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 according to OECD guideline 408 under GLP conditions (Sasol, 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.

One male in the mid-dose group was found dead on Day 75 of the study. This animal was seen with slight salivation at 0 – 1-hour post-dose on Days 14 - 16 and Day 57, but no other clinical signs were observed. At necropsy, macroscopic and microscopic findings in the liver were consistent with a spontaneous liver lobe torsion. The death is considered to be unrelated to the treatment.

During the dosing period, salivation was observed on several occasions shortly after dosing in 3/10 males and 1/10 females in the low-dose group. In total 9/10 males and 8/10 females were affected in the mid-dose group, and all males and females in the high-dose group, while no control animals were affected. No salivation was noted during the recovery period. The salivation was not considered toxicologically relevant and was seen as a physiological response due to gavage rather than a sign of systemic toxicity. Abnormal breathing sounds were noted in 1/10 mid-dose males on a single day, and in 10/15 males and 8/15 females in the high-dose group on several days during the dosing period. During the recovery period 1/5 males showed abnormal breathing sounds. Erected fur was observed once or a few times during the dosing period in 0/15, 1/10, 1/10 and 4/15 males in the control, low- mid- and high-dose group, respectively, and in 0/15, 0/10, 0/10 and 1/15 females in the control, low- mid- and high-dose group, respectively. Based on the low occurrence and short duration, these observations were not considered to be toxicologically relevant. No toxicologically relevant clinical signs were noted during weekly arena observations.

For males in the high-dose group, the body weight gain was significantly lower than controls from Day 57 onwards, resulting in a 12% lower mean body weight compared with the control at the end of the dosing period (Day 91). During the recovery period, a trend towards higher body weight gain was noted for high-dose males, resulting in a 5% lower mean body weight than for the controls (not statistically significant) at the end of the recovery period. Slightly higher body weight gain was observed in high-dose females compared to control throughout the treatment period, with a statistically significant increase in body weight of 6-7% compared with the control as measured on Day15, 22, 29 and 85 of the treatment. The changes in body weight gain in males and females were not considered related to treatment with the test substance.

Main groups: The neutrophil (NEUT) and monocyte (MONO) counts were significantly increased in high-dose males (1.44 and 1.64 x of control, respectively), causing a slight increase in white blood cell count (WBC; 1.20 x, not statistically significant). The red blood cell (RBC) count was significantly increased in high-dose males (1.05 x) and mid- and high-dose females (1.10 x for both). In males this increase in RBC count was caused by a slightly higher reticulocyte (RETIC) count (1.13 x, not statistically significant). Furthermore, in mid- and high-dose females a statistically significant increase in hemoglobin (HGB; 1.07 and 1.08 x, respectively) and hematocrit (HCT; 1.09 and 1.10 x, respectively) was observed. The platelet count (PLT) was significantly increased in mid- and high-dose females only (1.24 and 1.18 x, respectively). At the end of the recovery period, all haematological parameters in males and most parameters in females were similar to those of the respective control. Only HGB remained increased in high-dose females (1.04 x). Based on the reversibility, type of change (i.e. higher HGB), the low severity and/or in absence of a histopathological correlation, the listed changes in haematology parameters were considered to be non-adverse. The activated partial thromboplastin time (APTT) was shorter in high-dose females than for the control (0.88 x of control). At the end of the recovery period, APTT values were comparable to controls. In absence of a histopathological correlation, this finding was considered to be non-adverse.

Changes in clinical chemistry parameters at the end of the dosing period comprised an increase in alanine aminotransferase (ALT) activity in low-, mid- and high-dose males (1.24, 1.16 and 1.35 x of control, respectively; statistically significant at the low- and high dose level), increased aspartate aminotransferase (AST) in low-, mid- and high-dose males (1.29, 1.23 and 1.48 x, respectively; statistically significant at the high dose level) and alkaline phosphatase (ALP) activity in high-dose males and females (1.30 and 1.32 x, respectively; statistically significant only in males). In the recovery groups, the AST levels were significantly increased in high-dose females only (1.07 x). These changes are associated with the increased metabolic load on the liver of the treatment. The urea level at the end of the dosing period was significantly increased in low-, mid- and high-dose males (1.36, 1.52 and 1.61 x, respectively) and low-, mid- and high-dose females (1.21, 1.32 and 1.46 x, respectively). In low-, mid- and high-dose males, a significantly decreased level of cholesterol (0.85x, 0.77x and 0.73 x, respectively), HDL cholesterol (0.86, 0.78 and 0.68 x, respectively) and triglycerides (0.61, 0.56 and 0.48 x, respectively) was observed. The levels of urea, cholesterol, HDL cholesterol and triglycerides in the recovery group was similar to the control levels.

The thyroxine (T4) level were statistically significantly decreased in high-dose males of the main group (0.80 x), while the triiodothyronine (T3) concentration was significantly decreased in high-dose females (0.77 x). The thyroid stimulating hormone (TSH) concentration was increased in males and females (1.85 and 1.65 x, respectively), however, this was not statistically significant. After the recovery period, the TSH level was significantly decreased in high-dose males (0.26 x) while this was increased at the end of the dosing period (1.85 x), indicating an incidental change. All other values returned to normal values for both males and females. No histopathological alterations were observed in the thyroid, and the changes in thyroid hormone levels may be a secondary effect of the increased metabolic activity in the liver. The changes in thyroid hormone levels are therefore not considered to be toxicologically relevant.

A significant decrease in grip strength in the fore and hind legs was observed in high-dose males only (0.78 and 0.71 x, respectively) at the end of the dosing period. At the end of the recovery period, grip strength had recovered and was similar between high-dose males and control. For the main groups, a decreased number of total movements was observed: 0.68, 0.84 and 0.53 x for low-, mid- and high-dose females, respectively, compared with control (statistically significant for low- and high-dose). A decreased number of ambulations was also observed: 0.63, 0.87 and 0.54 x for low-, mid- and high-dose females, respectively, compared with control (statistically significant for low- and high-dose). At the end of the recovery period, a decreased number of total movements and ambulations was still present in high-dose females (0.72 x) compared to controls. The number of total movements and ambulations was slightly increased at the end of recovery period for high-dose males, compared to controls. Based on the absence of a dose-response and the opposite effect observed in males and females, this change was regarded to be not treatment-related. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

In the main group, increased liver weights were noted in all treatment groups, compared with the controls. The increase in relative liver weight was statistically significant in low-, mid- and high-dose males (8, 19 and 43%, respectively) and in mid- and high-dose females (18 and 49%, respectively). The increase in absolute liver weight was statistically significant in high-dose males (21%) and in mid- and high-dose females (21 and 55%, respectively). 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 mid- and high-dose females (16 and 22%, respectively) of the main group, compared with the control. The increase in relative kidney weight was statistically significant in mid- and high-dose males (13 and 20%, respectively) and in mid- and high-dose females (13 and 18%, respectively). Following the recovery period there was no difference in kidney weight in either sex. This change was not considered to be relevant as it was reversible and no histopathological changes were observed.

Prominent lobular architecture of the liver was recorded in 0/10, 3/10, 3/10 and 2/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 2/10 females in the control, low- mid- and high-dose group, respectively. As microscopic findings were noted at the high-dose level, the macroscopic findings at this dose level may be treatment-related. One of five (1/5) high-dose males in the recovery group showed prominent lobular architecture of the liver.

In the stomach, dark red foci in the glandular mucosa was noted in 0/10, 1/10, 0/10 and 5/10 males in the control, low- mid- and high-dose group, respectively. Microscopic lesions were also noted in high-dose animals. Irregular surface of the non-glandular stomach was noted in 0/10, 0/10, 0/10 and 2/10 males in the control, low- mid- and high-dose group, respectively. There were no macroscopic findings in the stomach of male or female animals at the end of the recovery period.

Treatment-related microscopic findings were noted in the thyroid gland, liver, jejunum, and stomach of males and females, and in the kidney of males.

Centrilobular hepatocellular hypertrophy was noted in 0/10, 0/10, 0/10 and 5/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 5/10 females in the control, low- mid- and high-dose group, respectively, following the dosing period. The effect is likely correlated to higher liver weights and to the prominent lobular architecture noted macroscopically. Centrilobular hypertrophy was not observed in males or females after the recovery period. The recovery indicates the effect may be caused by an increase in metabolic load following treatment.

A combination of findings was noted in the non-glandular stomach (forestomach) following the dosing period. Squamous cell hyperplasia (minimal to mild) was observed in 0/10, 0/10, 1/10 and 10/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 1/10 and 7/10 females in the control, low- mid- and high-dose group, respectively. Minimal erosion was noted in 1/10 males and 2/10 females in the high-dose group only. Submucosal edema (minimal to mild) was observed in 0/10, 0/10, 1/10 and 5/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 1/10 and 3/10 females in the control, low- mid- and high-dose group, respectively. In the non-glandular stomach mixed-cell submucosal infiltration (minimal) was observed in 0/10, 0/10, 2/10 and 3/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 2/10 and 2/10 females in the control, low- mid- and high-dose group, respectively.

In the glandular stomach, focal acute mucosal haemorrhage was noted in 0/10, 0/10, 1/10 and 3/10 males in the control, low- mid- and high-dose group, respectively. Mixed-cell submucosal infiltration of the glandular stomach (minimal) was noted in 1/10 males and 2/10 females in the high-dose group only.

The few occurrences in the low-, and mid-dose groups were not considered to be clear indications of a treatment-related effect. Following the recovery period, no lesions were noted in the stomach of the control animals. In the high-dose group, minimal squamous cell hyperplasia in the non-glandular stomach was noted in 1/5 females. Minimal vacuolation of the limiting ridge was noted in 1/5 males. Due to the correlated macroscopic and microscopic findings in the forestomach, these are considered toxicologically relevant in the high-dose group.

Vacuolation of the villous lamina propria of the jejunum was observed following the dosing period in 0/10, 0/10, 1/10 and 10/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 3/10 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. At the end of the recovery period, the same finding was made in 4/5 males and 5/5 high-dose females, respectively, and in no control animals. The results suggest that no recovery had occurred. However, 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 4/10, 4/10, 3/10 and 9/10 males in the control, low- mid- and high-dose group, respectively. The severity increased with dose, with most of the control males showing minimal accumulation and the high-dose animals showing mild to moderate accumulation. 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 2/10, 2/10, 3/10 and 7/10 males in the control, low- mid- and high-dose group, respectively, and in 2/10, 0/10, 1/10 and 6/10 females in the control, low- mid- and high-dose group, respectively. Following the recovery period, follicular cell hypertrophy was noted in 1/5 and 2/5 males in the control and high-dose group, respectively, and in 0/5 and 1/5 females in the control and high-dose group, respectively. This indicates a (partial) recovery of the microscopic thyroid gland effects.

A nephroblastoma was recorded in a high-dose male. This finding correlated with the macroscopic observation of a tan gelatinous content in the kidney. The tumour was located within the renal pelvis and most likely originated at this region. This was characterized by the presence of highly basophilic, densely packed, blast cells. Although the location of this particular tumour is unusual, this observation was interpreted to be a spontaneous change and not treatment-related given the single incidence in this study.

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

Studies in the AE category

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

Table 1

CAS No.

EC No.

Substance

Subgroup

Study protocol

Hazard conclusion

26183-52-8

500-046-6

Decan-1-ol, ethoxylated

Linear

OECD 422

NOAEL systemic ≥ 950 mg/kg bw/day

68439-50-9

500-213-3

Alcohols, C12-14, ethoxylated

Linear

OECD 422

NOAEL systemic ≥ 1000 mg/kg bw/day

9004-95-9

939-518-5

Hexadecan-1-ol, ethoxylated

Linear

OECD 422

NOAEL systemic ≥ 1000 mg/kg bw/day

NOAEL local = 300 mg/kg bw/day

68439-49-6

939-518-5

Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO

Linear

OECD 422

NOAEL systemic ≥ 1000 mg/kg bw/day

9004-98-2

500-016-2

(Z)-9-Octadecen-1-ol ethoxylated

Linear

OECD 422

NOAEL systemic ≥ 1000 mg/kg bw/day

160901-09-7

500-446-0

Alcohols, C9-11, branched and linear, ethoxylated

Mixed branched & linear

OECD 422

NOAEL systemic = 300 mg/kg bw/day

160901-19-9

500-457-0

Alcohols, C12-13, branched and linear, ethoxylated

Mixed branched & linear

OECD 422

NOAEL systemic = 300 mg/kg bw/day

106232-83-1

500-294-5

Alcohols, C12-15, branched and linear, ethoxylated

Mixed branched & linear

OECD 422

NOAEL systemic ≥ 1000 mg/kg bw/day

68439-50-9

500-213-3

Alcohols, C12-14, ethoxylated

Linear

OECD 408

NOAEL systemic ≥ 1000 mg/kg bw/day

68920-66-1

500-236-9

Alcohols, C16-18 and C18-unsatd., ethoxylated

Linear

OECD 408

NOAEL systemic = 300 mg/kg bw/day

160901-09-7

500-446-0

Alcohols, C9-11, branched and linear, ethoxylated

Mixed branched & linear

OECD 408

NOAEL systemic = 300 mg/kg bw/day

NOAEL local = 300 mg/kg bw/day

106232-83-1

500-294-5

Alcohols, C12-15, branched and linear, ethoxylated

Mixed branched & linear

OECD 408

NOAEL systemic ≥ 1000 mg/kg bw/day

NOAEL local = 300 mg/kg bw/day

The data available for alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) is consistent with the overall RDT data for AE substances. For a detailed evaluation of the repeated dose toxicity potential of the substances in the AE category, please refer to the category justification attached to the category object. 

Justification for classification or non-classification

The available subacute and subchronic data on repeated dose toxicity obtained with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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.