Hexadecan- l-ol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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

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

Oral (rat, m/f, OECD 422): NOAEL (local toxicity) ≥1000 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 toxicity to reproduction in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

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

Vehicle:

corn oil

Details on 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

Details on mating procedure:

Mating procedure
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

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.

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
On PND 4, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.


GROSS EXAMINATION OF DEAD PUPS
Pups found dead were examined for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

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

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

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

Postmortem examinations (offspring):

SACRIFICE
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two
surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two or three pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

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

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 were 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.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100

Offspring viability indices:

For each group, the following calculations were performed. Group mean values of duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

Gestation index (%):Number of females with living pups on Day 1/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%):Total number of offspring born/Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/
Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x100

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

Food consumption and compound intake (if feeding study):

no effects observed

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.

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:

not specified

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

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.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

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.
Most females had regular cycles of 4 to 5 days. An acyclic cycle was noted for a control female and a female receiving 300 mg/kg bw/day during the premating period, which both littered normally. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was not affected by treatment with the test item up to 1000 mg/kg bw/day. All paired females showed evidence of mating, which resulted in a mating index of 100% for all groups.
Precoital time was not affected by treatment with the test item up to 1000 mg/kg bw/day. All paired females showed evidence of mating within 6 days, except for one female receiving 300 mg/kg bw/day for which mating took 14 days. Given the single occurrence and in the absence of a dose-related trend, this longer mating period was considered not related to treatment with the test item.
Number of implantation sites was not affected by treatment with the test item up to 1000 mg/kg bw/day.
The mean number of implantation sites were 12.0, 12.3, 11.6 and 11.4 for the control, 100, 300 and 1000 mg/kg bw/day groups.
Fertility index was not affected by treatment with the test item up to 1000 mg/kg bw/day.
The fertility indices were 90, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One control female was not pregnant and was therefore not related to treatment with the test item.
Gestation index and duration of gestation were considered not to be affected by treatment
with the test item up to 1000 mg/kg/day.
The gestation indices were 100, 90, 100 and 100% for the control, 100, 300 and
1000 mg/kg/day groups, respectively.
The failed pregnancy of one female at 100 mg/kg/day, without related histopathology
changes in reproductive organs, was judged to be unrelated to treatment due to the incidental
occurrence.
No signs of difficult or prolonged parturition were noted among the pregnant females.

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:

histopathology: non-neoplastic

Remarks on result:

other: based on the microscopic findings in the non glandular stomach (forestomach) in males at 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:

reproduction

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 1000 mg/kg bw/day.
Clinical signs observed were:
One pup from control group pale on Day 4
One pup at 100 mg/kg bw/day with black tail at apex on first litter check and then missing tail on Day 13
One pup at 300 mg/kg bw/day with blue spot on neck on day of necropsy
One pup at 1000 mg/kg bw/day with wound to front leg on Day 6 was euthanized in extremis
One pup at 1000 mg/kg bw/day dehydrated with no milk in stomach on Day 1

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 1000 mg/kg/day.
The live birth index was 100% for all groups. One pup of the control group, one pup at 100 mg/kg/day, one pup at 300 mg/kg/day and one pup at 1000 mg/kg/day were missing on PND 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One female at 1000 mg/kg/day had a total litter loss on Lactation Day 2 as it only had one pup which was dehydraded and had no milk in its stomach on PND 1 and went missing on PND 2. At the incidence observed, no toxicological relevance was attributed to the total litter loss.
One pup at 1000 mg/kg/day was euthanized in extremis on PND 6 as it had a wound on its left front leg. No toxicological relevance was attributed to this mortality, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 in male and female 14-16 pups were considered not to be affected by treatment with the test item up to 1000 mg/kg/day.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Mean (normalized) anogenital distance of female pups was decreased at 300 and 1000 mg/kg/day (not always statistically significant) was not considered toxicologically relevant. Mean (normalized) anogenital distance of male pups was not affected with treatment with the test item up to 1000 mg/kg/day.

Nipple retention in male pups:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment up to 1000 mg/kg/day. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

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

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

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 8.7, of the REACH Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on toxicity to reproduction (fertility) 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)

The reproductive toxicity of hexadecan-1-ol, ethoxylated (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 relevant for reproductive toxicity were evaluated: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care. In addition, a number of developmental parameters as well as endpoints indicative of repeated dose toxicity were investigated. The details of the assessment of developmental toxicity are summarised below under 'Additional information' in the section for effects on developmental toxicity and the repeated dose toxicity details are provided in IUCLID section 7.5.1.

The length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item. Most females had regular cycles of 4 to 5 days. An acyclic cycle was noted for one female in the control and the mid-dose group (300 mg/kg bw/day) each during the premating period, which had littered normally. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment with the test item. The mating index was not affected by treatment as all paired females showed evidence of mating, which resulted in a mating index of 100% for all groups. Furthermore, the precoital time was not affected by treatment. All paired females showed evidence of mating within 6 days, except for one female in the mid-dose group (300 mg/kg bw/day) for which mating took 14 days. Since this was a single occurrence and in the absence of a dose-related trend, this longer mating period was considered not related to treatment. The number of implantation sites was also not affected by treatment with the test item. The mean number of implantation sites were 12.0, 12.3, 11.6 and 11.4 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Similarly, the fertility index was not affected by treatment. The fertility indices were 90, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One female in the control group was not pregnant. As this occurred in the control group, this was not related to treatment with the test item. Moreover,  the gestation index and duration of gestation were considered not to be affected by treatment because the gestation indices were 100, 90, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The failed pregnancy of one female in the low-dose group (100 mg/kg bw/day), without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

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

 

Studies in the AE category

Studies investigating toxicity to reproduction are available for the following AE substances:

Table 1

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

Conclusion on toxicity to reproduction (fertility)

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

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

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

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

 

For a detailed evaluation of the toxicity to reproduction potential of the substances in the AE category, please refer to the category justification attached to the category object.

Effects on developmental toxicity

Description of key information

Oral (rat, OECD 422): NOAEL (developmental toxicity) ≥ 1000 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 developmental toxicity in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Effect on developmental toxicity: via oral route

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 8.7, of the REACH Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on developmental 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)

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of hexadecan-1-ol, ethoxylated (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). The experimental details of the study are summarised above and in IUCLID section 7.5.1. Only information relevant for developmental toxicity are provided here. The following parameters relevant for developmental toxicity were evaluated: sex ratio and early postnatal pup development, i.e. mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, and measurement of thyroid hormone T4 (post-natal day (PND) 14-16).

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index was 97, 85, 91, 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The number of pups were slightly higher than the number of implantations for one female in the low-dose group (100 mg/kg bw/day). This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 14-16 days of) lactation. No toxicological relevance was attached to this finding in the current study. Litter size was also not affected by treatment and live mean  litter sizes were 11.7, 11.7, 10.6 and 10.1 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The slightly smaller mean litter sizes at 300 and 1000 mg/kg bw/day can be attributed to the smaller litter size of a single litter in each group. Litter Nos. 62 (300 mg/kg bw/day) and 78 (1000 mg/kg bw/day) consisted of 3 or 1 pup(s), respectively, at first litter check. In absence of a dose-related effect, these findings were considered not to be toxicologically relevant. The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. The live birth index was 100% for all groups. Furthermore, the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment; the viability index was 99% for all groups. One pup of the control group, one pup at 100 mg/kg bw/day, one pup at 300 mg/kg bw/day and one pup at 1000 mg/kg bw/day were missing on PND 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. One female in the high-dose group (1000 mg/kg bw/day) had a total litter loss on Lactation Day 2 as it only had one pup which was dehydrated and had no milk in its stomach on PND 1 and went missing on PND 2. At the incidence observed, no toxicological relevance was attributed to the total litter loss. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment with the test item up to 1000 mg/kg bw/day. The lactation indices were 100, 100, 100 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

One pup at 1000 mg/kg bw/day was euthanized in extremis on PND 6 as it had a wound on its left front leg. No toxicological relevance was attributed to this mortality, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. No clinical signs occurred among pups that were considered to be related to treatment with the test item. Body weights of pups and the sex ratio were also considered not to be affected by treatment. A slightly skewed sex ratio (60% males / 40% females) was recorded at 300 mg/kg bw/day, however, this was considered to be unrelated to treatment as it occurred in the absence of a dose-related trend and the values were within normal limits. The mean (normalised) anogenital distance of female pups was decreased at 300 and 1000 mg/kg bw/day. Only the mean normalised anogenital distance at 1000 mg/kg bw/day was statistically significantly lower compared to the concurrent control. Moreover, all mean values were within the historical control range. Therefore, this finding is not considered toxicologically relevant. The mean (normalised) anogenital distance of male pups as well as areola/nipple retention were not affected by treatment with the test item. For none of the examined male pups nipples were observed at PND 13 which was not considered toxicologically relevant. The serum levels of total T4 in male and female 14-16 pups were considered not to be affected by treatment. Finally, no macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item. No indication of a teratogenic effect of the test item was observed.

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

Studies in the AE category

Studies investigating toxicity to developmental toxicity are available for the following AE substances (Table 1-3):

Table 1: Overview of OECD 422 studies, developmental toxicity

CAS No.	EC No.	Substance	Screening study (OECD 422)
			NOAEL developmental (F1) [mg/kg bw/day]	NOAEL systemic (F0) [mg/kg bw/day]
Linear subgroup
26183-52-8	500-046-6	Decan-1-ol, ethoxylated	≥ 950	≥ 950
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	≥ 1000	≥ 1000
9004-95-9	939-518-5	Hexadecan-1-ol, ethoxylated	≥ 1000	≥ 1000 (local: 300)
68439-49-6	939-518-5	Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO	≥ 1000	≥ 1000
9004-98-2	500-016-2	(Z)-9-Octadecen-1-ol ethoxylated	≥ 1000	≥ 1000
Mixed branched & linear subgroup
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	300	300
160901-19-9	500-457-0	Alcohols, C12-13, branched and linear, ethoxylated	300	≥ 1000
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	300	≥ 1000

 

Table 2: Overview of OECD 414 studies in the rat

CAS No.	EC No.	Substance	Prenatal developmental toxicity study (OECD 414) in the rat
			NOAEL [mg/kg bw/day]
			Systemic (maternal)	Development	Teratogenicity
Linear
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	300	300	≥ 1000
68920-66-1	500-236-9	Alcohols, C16-18 and C18-unsatd., ethoxylated	1000	1000	1000
Mixed linear & branched
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	800	800	800
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	1000	1000	1000

 

Table 3: Overview of OECD 414 studies in the rabbit

CAS No.	EC No.	Substance	Prenatal developmental toxicity study (OECD 414) in the rabbit
			NOAEL [mg/kg bw/day]
			Systemic (maternal)	Development	Teratogenicity
Linear
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	30	200	200
68920-66-1	500-236-9	Alcohols, C16-18 and C18-unsatd., ethoxylated	(study ongoing)	-	-
Mixed linear & branched
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	100	400	400

 

Conclusion on developmental toxicity

All the data on developmental toxicity from the combined repeated dose toxicity study with the reproduction / developmental toxicity screening tests (OECD 422) and the prenatal developmental toxicity studies (OECD 414) in a rodent (rat) and a non-rodent species (rabbit) give a consistent picture of the effects across the category and species. Treatment with AE substances did not lead to adverse effects on most developmental parameters, including litter size, sex ratio, anogenital distance, placental weights of live foetuses and early postnatal offspring development consisting of mortality, clinical signs, areola/nipple retention, and macroscopic examination. In two studies, performed with substances in the mixed branched & linear subgroup, reduced offspring body weight was observed at a dose inducing significant maternal toxicity and this was considered a secondary effect of the maternal toxicity. In one study, performed with a substance in the mixed branched & linear subgroup (alcohols, C12-15, branched and linear, ethoxylated) reduced offspring body weight was observed at the highest dose level without clear maternal systemic toxicity. As this was the only study among the eight studies in which an effect was observed on the offspring generation only, this is considered specific to this substance and not relevant to the category as a whole. No teratogenic effects were observed. There was no clear difference in (lack of) reproductive and developmental and effects between the linear subgroup and the mixed branched & linear subgroup, indicating consistency across the category.

The data on developmental toxicity and teratogenicity available for hexadecan-1-ol, ethoxylated (CAS No. 9004-95-9, EC No. 500-014-1) is consistent with the overall toxicity to reproduction data for AE substances. The following NOAEL was set:

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

For a detailed evaluation of the toxicity to reproduction 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 data on toxicity to reproduction 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.