Hexadecan- l-ol, ethoxylated

Endpoint summary

Description of key information

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

Oral (subacute, rat, m/f, OECD 422): NOAEL (local toxicity) = 300 mg/kg bw/day

Conclusion based on data obtained with hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1) 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

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jul 2020 - 06 Apr 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 299-347 g, females 186-225 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: 6 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 52-76
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From: 14 JUL 2020 to 16 SEP 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.
- The dosing formulations were prepared weekly as a solution through heating to a maximum temperature of 40 ± 1°C under continuous stirring for at least 30 minutes to obtain visual homogeneity.
-Test item dosing formulations were kept at 40 ± 1°C until and during 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 (Test Facility Study No. 20243920) 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 (Test Facility Study No. 20243920).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was less than or equal to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

Duration of treatment / exposure:

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

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a 10 day Dose Range Finder in rats (Test Facility Reference No. 20243922), and in an attempt to produce graded responses to the test item.
F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

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

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

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

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

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

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

Sacrifice and pathology:

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

GROSS 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 according when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

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

Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related salivation was noted during daily detailed clinical observations starting at 100 mg/kg bw/day in males and at 300 mg/kg bw/day in females in a dose-related manner.
Incidental findings that were noted included alopecia, rales and piloerection. 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.
No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was related to treatment with the test item up to 1000 mg/kg bw/day.
One female from the control group was euthanized in extremis on Day 2, as it had broken its left front paw after jumping out of its cage. As this accidental injury occurred during the replacement period, this female was replaced by an alternate female.
One female at 1000 mg/kg bw/day was scheduled for euthanasia on Lactation Day 2, as it had a total litter loss.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item 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 up to 1000 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were affected by treatment with the test item at 100, 300 and 1000 mg/kg bw/day with the following statistically significant changes distinguished treated from control animals:
• Mean glucose concentration was increased (1.40x, 1.26x and 1.27x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean cholesterol concentration was increased (1.36x of control) at 1000 mg/kg bw/day in males.
• Mean potassium concentration was increased (1.10x, 1.08x and 1.09x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean bile acid concentration was increased (1.57x and 2.09x of control) at 300 and 1000 mg/kg bw/day in females, respectively.
Mean values, including potassium concentration in treated males and bile acid concentration in females , achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution or corroborative findings in the opposite sex were therefore considered to be non-adverse and of no toxicological significance.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item due to the minimal magnitude of the change and/or absence of a dose-related response

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum levels of total T4 was statistically significantly decreased at 1000 mg/kg bw/day in F0 males, but not in F0 females.
Mean serum levels of total TSH was statistically significantly increased at 300 mg/kg bw/day in F0 males. In the absence of a dose-related trend, this change was considered to be unrelated to treatment with the test item.
The increase in mean serum levels of TSH in F0 females at 300 and 1000 mg/kg bw/day was considered a result of one or two outliers and was therefore considered to be of no toxicological relevance. In the absence of a dose-related trend, the increase at 100 mg/kg/day was considered to be of no toxicological relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment up to 1000 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength and motor activity was similar between control and high dose animals.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A test item-related increase in adrenal gland weights was noted at 1000 mg/kg bw/day in males as shown in Table 1 and possible test item-related increased liver weights were noted at 1000 mg/kg bw/day in females as shown in Table 2. These findings were considered as non-adverse since these changes were not associated with any adverse pathological alterations.
Any other differences, including those that reached statistical significance (relative kidney weights at 1000 mg/kg bw/day in males) were considered not to be related to treatment with the test item due to the direction of the change, lack of dose-related pattern, absence of microscopic correlate and/or general overlap and variability in individual values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the non-glandular stomach and pituitary gland at 1000 mg/kg bw/day in males and in the jejunum at 300 and 1000 mg/kg bw/day in both sexes. These findings are summarized in Table 3 and Table 4.

Non-glandular stomach (forestomach): Squamous cell hyperplasia of the non-glandular epithelium was observed at a minimal degree in 3/5 males at 1000 mg/kg bw/day. This finding was in two males accompanied by (sub)mucosal (lympho)granulocytic infiltrate, and in one of the two also by a slight ulcer of the epithelium. These findings were considered adverse.

Pituitary gland: an increased incidence and severity of hypertrophy and vacuolation of cells of the pars distalis was observed at 1000 mg/kg bw/day in 3/5 males (up to slight degree). These were considered to be non-adverse test item-related morphologic alterations.

Jejunum: Lymphangiectasis (i.e. dilated lacteals in the villi) was noted in 1/5 males and 3/5 females at 300 mg/kg bw/day (up to a slight degree) and in 4/5 males and 5/5 females at 1000 mg/kg bw/day (up to moderate degree). These were considered to be non-adverse test item-related morphologic alterations.

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.

Other effects:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
The stage dependent qualitative evaluation of spermatogenesis in the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

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

Dose descriptor:

NOAEL

Remarks:

Local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant efects observed

Key result

Dose descriptor:

LOAEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

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 study is 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 hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Study with hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1)

Hexadecan-1-ol, ethoxylated, < 2.5 EO (CAS No. 9004-95-9, EC No. 500-014-1) 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 (CRL, 2021). Groups of 10 animals per sex received doses of 100, 300 and 1000 mg/kg bw/day by daily oral gavage, 7 days a week for a minimum of 28 days. A similarly constituted control group was dosed with the vehicle (corn oil) only. Males were treated for 29 days whereas females that delivered were treated for 50-64 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver or had a total litter loss were treated for 40-43 days. The following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations. In addition, a number of reproduction / developmental parameters were investigated. The details of the reproductive/developmental screening test are summarised in IUCLID section 7.8.1.

One female from the control group was euthanised in extremis on Day 2, as it had broken its left front paw after jumping out of its cage. As this accidental injury occurred during the replacement period, this female was replaced by an alternate female. Salivation seen after dosing among animals at 100, 300 and 1000 mg/kg bw/day was considered non-adverse, taking into account the nature and minor severity of the effects and its time of occurrence after dosing. This observation was considered to be a physiological response rather than a sign of systemic toxicity. Changes observed in clinical chemistry parameters (increased plasma glucose starting at 100 mg/kg bw/day and cholesterol at 1000 mg/kg bw/day in males) and organ weights (increased adrenal gland weights at 1000 mg/kg bw/day in males and increased relative liver weights at 1000 mg/kg bw/day in females) were considered non-adverse since these changes were not associated with any adverse pathological alterations. A test item-related decrease in serum total T4 concentration was noted for F0-males at 1000 mg/kg bw/day. No adverse effect was observed that could be linked to this reduction of total T4 and therefore the reduction was not considered adverse. Local test item-related morphological alterations were present in the non-glandular stomach (forestomach) at 1000 mg/kg bw/day in males. The changes consisted of squamous cell hyperplasia and/or (lympho)granulacytic infiltrate. These findings together with ulceration found in a single male at 1000 mg/kg bw/day were considered adverse. Non-adverse test item-related morphologic changes were present in the jejunum (lymphangieactasis) starting at 300 mg/kg bw/day in both sexes and in the pituitary gland (hypertrophy and vacuolation of cells of the pars distalis) at 1000 mg/kg bw/day in males. Based on the findings of this study, No-Observed-Adverse-Effect-Levels (NOAEL) of ≥ 1000 mg/kg bw/day for parental systemic and 300 mg/kg bw/day for parental local toxicity were determined.

Studies in the AE category

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

Table 1

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

Evaluation of repeated dose toxicity as observed in available studies

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

The combined repeated dose toxicity study with the reproductive / developmental toxicity screening test was performed according to OECD guideline 422 under GLP conditions. Groups of 10 rats per sex received the test substance by daily oral gavage, 7 days a week for a minimum of 28 days. A similarly constituted control group was dosed with the vehicle (corn oil) only. The dose levels were set based on the guideline recommendation for substances not expected to exhibit strong systemic toxicity. Males were treated for 29 days whereas females that delivered were treated for 50-64 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females that failed to deliver or had a total litter loss were treated for 40-43 days. The following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations. In addition, a number of reproduction / developmental parameters were investigated.

The Repeated dose 90-day oral toxicity study in rodents was performed according to OECD guideline 408 under GLP conditions. Groups of 10 rats/sex were administered the test substance by oral gavage, 7 days a week for a minimum of 90 days. The control group was treated according to the same protocol and received the vehicle (corn oil) only. A satellite group of 5 animals/sex was included in the control and 800 mg/kg bw/day group to assess the recovery from any treatment-related effects. The dose levels were based on the results of the OECD 422 studies. Following the treatment period, the recovery period for the satellite animals was 28 days. The following parameters were recorded: mortality/moribundity, clinical signs, detailed clinical observations, body weight and food consumption, water consumption, opthalmoscopic examination, estrous cycle determination, sperm analysis, haematological parameters, clinical chemistry parameters, measurement of thyroid hormones (T3, T4 and TSH), urinalysis, neurobehavioural examination, gross necropsy, organ weights and histopathologic examination.

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

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

 

Clinical signs

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

 

Body weight development

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

 

Stomach

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

Gastrointestinal tract, jejunum

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

 

Liver

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

 

Lungs and airways

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

 

Sperm analysis

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

 

Oestrous cycle

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

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

Conclusion on RDT

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

The data available for hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1) is consistent with the overall RDT data for AE substances. The following NOAELs were set:

 

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

Oral (subacute, rat, m/f, OECD 422): NOAEL (local toxicity) = 300 mg/kg bw/day

 

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 hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1) 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.