(Z)-9-Octadecen-1-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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 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:

03 Dec 2019 - 17 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 in 2016

Deviations:

yes

Remarks:

please refer to "Principles of methods if other than guideline" for details

Principles of method if other than guideline:

Deviations to OECD 422 (2016): no functional observations and motor activity measurements were performed at the end of treatment for female animals, for one male, the testis and epididymides were directly placed in formalin instead of first modified Davidson's fixative for a minimum of 24 h; no food consumption was recorded for one female during LD 1-4, no bw and nipple retention were determined on PND13 for pups in two of the litters of the control group, it could not be retrieved from the raw data if one of the females dosed at 300 mg/kg bw/day was dosed post coitum Day 5; the fasting period exceeded 24 h in 4 males (3-64 min exceedance) of the 100 mg/kg bw/day group

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Limit test:

no

Justification for study design:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

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. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: Males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: Males 261-355g, females 203-246g
- 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): 22-22
- Humidity (%): 39-57
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From 12 FEB 2020 to 16 APR 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.
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until dosing.
- Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on 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 (UPLC-MS based on ammonium adduct).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20218716).

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. 20218717) with oral gavage administration of (Z)-9-Octadecen-1-ol ethoxylated (1.2 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.

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 males: Testis weight, epididymis weight, seminal vesicle weight was recorded. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed.

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 determined for pups born or found dead.

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.

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

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):

Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

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 pups per litter and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

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.

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

Gestation index (%): Number of females with living pups on Day 1 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.

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

For each group, the following calculations were performed:

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):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations.No findings were noted during the weekly arena observations in this study. Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups starting in Week 4 or Week 2 of treatment, respectively, was considered not toxicologically relevant, taking into account the nature and in general limited severity of the effect and its time of occurrence (i.e. after dosing). This observation was considered to be a physiological response rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment 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 low incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no mortalities that could be attributed to treatment with the test item.
One female of the control group was euthanized on lactation Day 2, as she had a total litter loss.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered not affected by treatment. In males at 1000 mg/kg bw/day, body weight gain was decreased with statistical significance on Day 8 of pre-mating. In females at 1000 mg/kg bw/day, body weight gain was increased with statistical significance on Day 7 post-coitum and Day 7 of lactation. These and any remaining statistically significant changes were considered not toxicologically relevant as no trend was apparent regarding dose and duration of treatment and mean values were within normal ranges of biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded. In females at 1000 mg/kg bw/day, absolute and relative food consumption on Days 1-8 of pre-mating were slightly lower (0.86x of control for absolute food consumption, not statistically significant). This change was considered treatment-related. However, as complete recovery of food consumption was recorded during Days 8-15 of treatment, no toxicological relevance was attributed to this finding.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters were considered not affected by treatment with the test item in males up to 1000 mg/kg bw/day.
Decreased white blood cell (WBC) counts was seen in females at 1000 mg/kg bw/day (0.69x of control), caused by a slight decrease in neutrophils, lymphocytes and eosinophils. This change was considered treatment-related. Increased red blood cell distribution width (RDW) was seen in females at 1000 mg/kg/day (1.12x of control). This change was considered treatment-related. Any other statistically significant changes in hematological parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change.
Coagulation parameters of treated rats were considered not affected by treatment with the test item in females up to 1000 mg/kg bw/day. Increased platelet counts in males at 300 and 1000 mg/kg bw/day (up to 1.21x of control) were observed, which were considered test item-related. Mean values remained within the historical control range. Decreased platelet counts in females at 100 mg/kg bw/day (0.76x of control) were recorded. As no dose-relation was apparent, this change was considered not toxicologically relevant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased alanine aminotransferase (ALAT) activity was seen in males at 1000 mg/kg/day (1.95x of control). Mean value was outside the historical control range. This change was considered treatment-related. Increased cholesterol levels was seen in males (1.21x of control) and females (1.18x of control, not statistically significant) at 1000 mg/kg bw/day. Mean values were outside the historical control range for males only. These changes were considered treatment-related. Increased bile acid concentrations was seen in males at 1000 mg/kg bw/day. Bile acids were increased as well in males at 300 mg/kg bw/day and in females starting at 100 mg/kg bw/day (not statistically significant). These changes were considered the result of slightly low control values and not related to treatment. Decreased aspartate aminotransferase (ASAT) activity was seen in males at 100 mg/kg bw/day and was considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Endocrine findings:

not specified

Description (incidence and severity):

Serum levels of T4 in F0-males were decreased starting at 100 mg/kg bw/day (up to 0.80x of control, statistically significant at 100 and 1000 mg/kg bw/day only). Mean values were within the historical control range and control values were relatively low. This effect was therefore considered not toxicologically relevant.

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 male animals up to 1000 mg/kg bw/day. Grip strength of the fore and hind legs were generally lower in males of all treated groups compared to the concurrent controls. It should be noted that the mean control values were at the higher end of the historical control range. As mean values of treated groups remained within historical control range2 and in the absence of a dose-related trend these decreases were considered unrelated to treatment with the test item. In males, a trend towards lower values of total movements and ambulations was observed at 1000 mg/kg bw/day (0.77x and 0.76x of control, respectively, not statistically significant), which was considered possibly 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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were test item-related microscopic observations in the jejunum of males and females treated at 1000 mg/kg bw/day, as summarized in Table 2. Vacuolation in the lamina propria of the jejunum was observed in 3/5 males (up to slight degree) and 4/5 females (up to moderate degree). Based on the absence of any additional degenerative or inflammatory changes the finding was considered non-adverse. All remaining 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

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not affected by treatment. All females had regular cycles of 4 days during pretest and premating. Extended di-estrous occurred in Female Nos. 42, 43 (Group 1), 63, 67, 68 (Group 3) and 77 (Group 4) during the mating period (all with normal litters). Given the absence of a dose-related incidence, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. 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):

There were 1/10 males of the control and 300 mg/kg bw/day group each that did not mate, 1/10 females of the control group with total litter loss, and 3/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. Mating index was not affected by treatment. All females showed evidence of mating. Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within 4 days. One control female and one female at 300 mg/kg bw/day mated on the first day of the second mating period. Extended di-estrous during the mating period was considered the cause of absence of mating during the first mating period. Number of implantation sites was considered not to be affected by treatment. The mean number of implantation sites was 11.7, 11.8, 12.6 and 12.9 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. All individual values remained within the normal range of biological variation. Fertility index was considered not to be affected by treatment. The fertility indices were 100, 100, 70 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. A total of three females at 300 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment. Gestation index and duration of gestation were considered not to be affected by treatment. 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.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

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

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

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. These were: scab on ear and abscess on head for one pup at 1000 mg/kg bw/day and blue spot on flank for a pup in the same litter. Alopecia for a number of pups from 2 litters at 100 mg/kg bw/day. The nature and incidence of clinical signs remained within the range considered normal for pups of this age.

Dermal irritation (if dermal study):

not examined

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 considered not to be affected by treatment. The live birth indices were 94, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Six pups of the control group and one pup at 100 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. Viability indices were 98, 99, 97 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100% for the control, 100, 300 mg/kg bw/day groups, respectively, and 99% for the 1000 mg/kg bw/day group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg bw/day/day had no effect on areola/nipple retention for none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings (other than those reported as clinical signs) were noted among pups and these were considered not to be related to treatment with the test item. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1: Mean Percent Liver Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg bw/day):	100		300		1000	100	300	1000
LIVER
Absolute	0		9		11	2	8	14*
Relative to body weight	3		10		15**	4	7	12**

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

 

Table 2: Summary Test Item-Related Jejunum Findings – Scheduled Euthanasia Animals

	Males				Females
Dose level (mg/kg bw/day):	0	100	300	1000	0	100	300	1000
JEJUNUM ^a	5	5	5	5	5	5	5	5
Vacuolation, lamina propria
minimal	-	-	-	1	-	-	-	2
slight	-	-	-	2	-	-	-	1
moderate	-	-	-	-	-	-	-	1

^a  =  Number of tissues examined fro

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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 

The reproductive toxicity of (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 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, 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 41-42 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.

All females had regular cycles of 4 days during pre-test and premating. Extended di-estrous occurred in two females of the control group, three females of the mid-dose group (300 mg/kg bw/day) and one female of the high-dose group (1000 mg/kg bw/day) during the mating period (all with normal litters). Given the absence of a dose-related incidence, these findings did not indicate a relation with treatment. Mating index was not affected by treatment. All females showed evidence of mating and the precoital time was considered not to be affected by treatment. Most females showed evidence of mating within 4 days. One control female and one female at 300 mg/kg bw/day mated on the first day of the second mating period. Extended di-estrous during the mating period was considered the cause of absence of mating during the first mating period. The number of implantation sites was considered not to be affected by treatment. The mean number of implantation sites was 11.7, 11.8, 12.6 and 12.9 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. All individual values remained within the normal range of biological variation. Furthermore, the fertility index was considered not to be affected by treatment. The fertility indices were 100, 100, 70 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. A total of three females at 300 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment. The mean duration of gestation was 21.4, 21.2, 21.0 and 21.1 days for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The gestation index was 100% for all groups. 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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) are 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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 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, 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. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 93, 84 and 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. For one female, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during (the 16 days of) lactation. No toxicological relevance was attached to this finding in the current study. Litter size was considered not affected by treatment. Live litter sizes at first litter check were 10.0, 10.9, 10.6 and 11.5 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The live birth indices were 94, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Six pups of the control group and one pup at 100 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. Viability indices were 98, 99, 97 and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One control female had total litter loss on PND 2 (six pups were found dead at first litter check and the two remaining pups were missing on PND 2). No clinical signs or body weight changes were observed, and no macroscopic or microscopic findings were recorded that could explain the total litter loss. Additionally, one pup of the 100 mg/kg bw/day group, two pups of the 300 mg/kg bw/day group, and one pup of the 1000 mg/kg bw/day group were found dead or missing on PND 2-3. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/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. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100% for the control, 100, 300 mg/kg bw/day groups, respectively, and 99% for the 1000 mg/kg bw/day group. One pup at 1000 mg/kg bw/day was sacrificed in extremis on PND 10 with an abscess on the head. This isolated finding was considered to be unrelated to treatment with the test item. No clinical signs occurred among pups that were considered to be related to treatment with the test item. The nature and incidence of clinical signs remained within the range considered normal for pups of this age. The body weights and sex ratio of pups were considered not to be affected by treatment with the test item. The anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item. Treatment up to 1000 mg/kg bw/day/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item. No macroscopic findings were noted among pups that were considered to be related to treatment with the test item. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age. 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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) 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 (Z)-9-octadecen-1-ol, ethoxylated (CAS No. 9004-98-2, EC No. 500-016-2) and with other members of the Alcohol Ethoxylates (AE) category do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.