Alcohols, C9-11, branched and linear, 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) = 300 mg/kg bw/day

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

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

 

Conclusion based on data obtained with alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) and considering all the available data on 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:

23 Jan - 12 Jun 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. 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: 11-13 weeks
- Weight at study initiation: 205-242 g
- Fasting period before study: NA
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days for males and 10 days for females

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

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

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

Route of administration:

oral: gavage

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 had 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.
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study (Test Facility Study No. 20219836).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 29 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were
treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of Alcohols, C9-11, branched and linear, ethoxylated in rats, and in an attempt to produce graded responses to the test item (Test Facility Reference No. 20219837).

- 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 all F0 males: 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 evaluation of external genitalia]

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.

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.

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

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

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

Postmortem examinations (offspring):

SACRIFICE
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.

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 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

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):

For all females treated at 1000 mg/kg/day, piloerection and slight to severe lethargy were noted during the entire study period. In the beginning of the study, the majority of the animals were affected and this was accompanied by abnormal (7/10), flat (9/10) and hunched posture (10/10) and/or uncoordinated movements (9/10). At the end of study, these clinical signs were noted in individual animals and (ventro-)lateral recumbency (1/10), loss of righting reflex (1/10), decreased locomotor activity (2/10) and/or abnormal gait (1/10) were also noted in individual animals. Rales (6/10) was noted for the majority of females during the entire study period, which was incidentally accompanied with slow breathing (2/10), labored (1/10), deep (1/10) and/or shallow respiration (2/10) for individual animals. Other clinical signs that were incidentally noted for individual animals included chromodacryorrhoea (1/10), lean appearance (2/10) and ptosis (3/10). For males treated at 1000 mg/kg/day, rales (8/10) was noted for the majority of the animals during the last two weeks of the study, which was accompanied by labored respiration (2/10) in individual animals. Piloerection (1/10) was also incidentally noted for individual animals. At the incidence observed, these clinical signs were considered not toxicologically relevant. At 300 mg/kg/day, rales and chromodacryorrhoea were incidentally noted in males and piloerection was incidentally noted in females, based on the incidence observed, these clinical signs were considered not toxicologically relevant. Salivation seen after dosing for males and females at 1000 mg/kg/day and males at 300 mg/kg/day was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

Endocrine findings:

not specified

Description (incidence and severity):

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

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the forestomach of both sexes and thyroid gland of males of the 1000 mg/kg/day group and are summarized in Table 2. At 1000 mg/kg/day, signs of local irritation of the forestomach (i.e. non-glandular stomach) were present in the form of squamous cell hyperplasia with hyperkeratosis and subepidermal lymphogranulocytic infiltrate in both sexes and additional sub(mucosal) edema in males. The macroscopic correlate for the hyperplasia and edema consisted of irregular surface of the forestomach and/or thickened limiting ridge. Since severities of these microscopic findings were low (up to slight degree) and were present only in the superficial layers of the forestomach, they were regarded as non-adverse. The test item-related increased incidence of follicular cell hypertrophy of the thyroid gland of 1000 mg/kg/day males was at the observed minimal severity and in absence of other test-item related thyroid gland changes regarded non-adverse.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. This included the requested liver of males, showing incidences of one control, one 100 mg/kg/day, zero 300 mg/kg/day and two 1000 mg/kg/day group males with minimal hepatocellular hypertrophy.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Normal length and regularity of the estrous cycle was disturbed in 5/10 females at 1000 mg/kg/day. This was considered related to treatment with the test item and at this incidence considered adverse. Despite the disturbance in estrous cycle during premating, all females showed evidence of mating within 4 days after the start of the mating period. It therefore was considered that the cohabitation with a male stimulated the females to a reset of their estrous cycle. Disturbance of the estrous had no effect on the mating performance and fertility as all female delivered offspring.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The testes 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 couples of the control group, 3/10 couples of the 100 mg/kg/day group and 2/10 couples of the 300 mg/kg/day group without offspring despite proof of mating. 1/10 males at 300 mg/kg/day failed to sire and 1/10 females at 100 mg/kg/day had total litter loss at lactation day 6. This lack of healthy offspring could not be explained by the microscopic examination of the reproductive organs. Mating index was not affected by treatment. All females showed evidence of mating. The mating indices were 100% for all groups. Pre-coital time was considered not to be affected by treatment. Number of implantation sites was considered not to be affected by treatment with the test item. Fertility index was considered not to be affected by treatment. The fertility indices were 90, 70, 90 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. 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.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

presumably yes

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. The nature and incidence of the clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not toxicologically relevantly affected. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 86, 87 and 82% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. Litter size was considered not affected by treatment.
Live litter sizes were 10.2, 10.7, 11.9 and 9.7 living pups/litter for the control, 100, 300 and 1000 mg/kg/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 97, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. 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 100, 100, 98 and 100% for the control, 100, 300 and 1000 mg/kg/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, 98, 98 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower body weights of pups at 1000 mg/kg/day were noted from PND 1 onwards (up to 0.85x and 0.87x of control in male and female pups, respectively; not always statistically significant). Other statistically significant changes in body weights of the pups were considered to be unrelated to treatment as these occurred in absence of a dose-related trend.

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/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):

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.

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:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Table 1
Mean Percent Liver Weight Differences from Control Groups

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

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

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

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

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

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus 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 alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

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

The reproductive toxicity of alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) was tested in Wistar Han rats in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test according to OECD guideline 422 under GLP conditions (Innospec, 2020). Groups of 10 animals per sex received doses of 100, 300 and 1000 mg/kg bw/day by daily oral gavage, 7 days a week for a minimum of 28 days. A similarly constituted control group was dosed with the vehicle (corn oil) only. Males were treated for 29-38 days whereas females that delivered were treated for 50-67 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver or had a total litter loss were treated for 42-43 days. The following parameters and endpoints 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.

Normal length and regularity of the estrous cycle was disturbed in 5/10 females at 1000 mg/kg bw/day: At 1000 mg/kg bw/day, 3/10 females were acyclic, 1/10 females had irregular cycles and for 1/10 females it was not possible to determine the length and regularity of the estrous cycle. This was considered related to treatment with the test item and at this incidence considered adverse. Despite the disturbance in estrous cycle during premating, all females showed evidence of mating within 4 days after the start of the mating period. It therefore was considered that the cohabitation with a male stimulated the females to a reset of their estrous cycle. Disturbance of the estrous had no effect on the mating performance and fertility as all females delivered offspring. All females of the control group and the 100 and 300 mg/kg bw/day group had regular cycles of 4 days. Mating index was not affected by treatment as all females showed evidence of mating. The mating indices were 100% for all groups. In absence of a dose-related trend this was considered not related to treatment with the test item. All females showed evidence of mating within 6 days, except for one animal of the control group and one animal treated at 100 mg/kg bw/day, which showed evidence of mating after 14 and 13 days, respectively. Hence, the precoital time was considered not to be affected by treatment. One female at 100 mg/kg bw/day, which had a total litter loss on PND 6, had three implantation sites. As this occurred in absence of a dose response relationship, this was considered to be unrelated to treatment. A total of one control female, three females at 100 mg/kg bw/day and one female at 300 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment with the test item. Fertility index was therefore considered not to be affected by treatment. The fertility indices were 90, 70, 90 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Except for one female at 300 mg/kg bw/day, all pregnant females had live offspring. The gestation indices were 100, 100, 89 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The failed pregnancy of the female, which had implantation only, was without related histopathology changes in reproductive organs and was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend. No signs of difficult or prolonged parturition were noted among the pregnant females. In conclusion, no toxicologically significant changes were noted in any of the other reproductive parameters investigated, i.e. mating and fertility indices, precoital time, number of implantations, spermatogenic profiling, and histopathological examination of reproductive organs.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of 300 mg/kg bw/day for reproductive toxicity was determined. It is important to note that the reproduction toxicity observed in this study occurred at dose levels associated with maternal systemic toxicity. This included clinical signs for females during the entire study period. It is reasonable to assume that these clinical signs were related to the observed reproduction toxicity. The disturbance of the oestrus cycle found in 5/10 females at 1000 mg/kg bw/day can therefore be regarded as a secondary effect to the systemic maternal toxicity. This conclusion is further supported by the fact that no reproduction toxicity was observed at dose levels which were non-toxic to the dams.

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 alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) is in general consistent with the overall toxicity to reproduction data for AE substances.In the study with alcohols, C9-11, branched and linear, ethoxylated a disturbed oestrus cycle was found in 5/10 females and judged to be adverse. However, this finding was not observed in any of the subchronic repeated dose toxicity studies and had no impact on the fertility of the females in this study. Therefore, it is considered an incidental effect of no toxicological relevance for this or any other AE category member substance.

The following NOAELs were set: 

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

Oral (rat, m/f, OECD 422): NOAEL (systemic toxicity) = 300 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 414): NOAEL (developmental toxicity) = 800 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 800 mg/kg bw/day

 

Conclusion based on data obtained with alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) and considering all the available data on developmental toxicity in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jul - 26 Aug 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 - 13 weeks
- Weight at study initiation: 183 - 258 g
- Fasting period before study: not applicable
- Housing: individually in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: 5 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 58 - 67
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Jul 2021 To: 26 Aug 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The appropriate amount of the test material was mixed with the vehicle. The dose volume for each animal was based on the most recent body weight measurement and the dose formulations were stirred continuously during dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analyses confirmed that formulations of the test item in corn oil were prepared accurately and homogenously. Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration).
No ammonium adduct of the C10E6 compound in the test item was detected in the Group 1 formulation.
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10 %).

Details on mating procedure:

- Impregnation procedure: not reported
Females were time-mated and arrived at the testing facility as such. Day 0 of post-mating (Day 0 of gestation) is the day of mating.

Duration of treatment / exposure:

Day 6 - 20 post-mating

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy on Day 21 post-mating

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

800 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels in this study were selected to be 100, 300 and 800 mg/kg bw/day, based on the results of a Combined 28-day Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test with Alcohols, C9-11, branched and linear, ethoxylated in rats (Test Facility Study No. 20219839), and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: Day 21 post-mating

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-mating up to the day prior to necropsy, morbidity and mortality were performed twice daily, observations were performed directly post dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-mating

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-mating.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-mating.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: on a regular basis throughout the study (monitored by visual inspection)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 post-mating
All animals from all groups were subjected to a gross necropsy. All gross lesions were collected.
- Organs weighed: thyroid
- Tissues collected for histopathology: thyroid gland and macroscopic abnormalities.
- Fixative: 10% buffered formalin
- Embedding media: paraffin
- Thickness of sections: 2 - 4 µm
- Staining: hematoxylin and eosin

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: 1.0 mL
- Parameters checked: triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH).

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes (all per litter)
- Body weight: Yes (all per litter)
- Sex: Yes (all per litter)

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. All pairwise comparisons were conducted against the control group (Group 1).

The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test, ANCOVA and Fisher's exact test.

Indices:

Pregnancy rate (%): (No. of pregnant females)/(No. of mated females) * 100
Male fetuses (%): (No. of male fetuses)/(No. of fetuses) * 100
Female fetuses (%): (No. of female fetuses)/(No. of fetuses) * 100
Pre-implantation loss (%): (No. of corpora lutea – No. of implantations)/(No. of corpora lutea) * 100
Post-implantation loss (%): (No. of implantations – No. of live fetuses)/(No. of implantations) * 100
Litter % of fetuses with abnormalities: (No. of fetuses in litter with a given finding)/(No. of fetuses in litter examined) * 100

Historical control data:

Historical control data was provided and can be found in Attachment 3 under "Overall Remarks, Attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Various clinical signs were observed throughout the treatment period. Abnormal breathing sounds observed in some animals of all treatment group were considered incidental as they occurred sporadically. Erected fur in 3/22 females of the 800 mg/kg bw/day group, 1/22 female of the 300 mg/kg bw/day and 1/22 female of the control group was also considered incidental as well as slight salivation seen in 11/22 and 3/22 females after dosing at 800 and 300 mg/kg bw/day, respectively, mostly on incidental occasions, with a maximum of 4 consecutive days. All clinical signs were considered treatment-related but not adverse.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

At 800 mg/kg bw/day, one female was sacrificed in extremis on Day 12 post-mating, as it was noted with labored breathing, lateral recumbent position, a weak appearance and decreased activity, cold to the touch and slight salivation after dosing. Slight body weight loss (3%) was noted between Days 6 - 9 post-mating, followed by no recovery thereafter. In addition, food consumption was lower between Days 6 - 12 post-mating (11 g/day vs. 20 g/day during the acclimatization period from Days 2 - 6 post-mating). At necropsy, no macroscopic alterations were found. This female was non-gravid. The death was considered treatment-related.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the low- and mid- dose group, no statistically significant difference in body weight was observed between control and treatment groups. In the high-dose group, a general trend towards slightly lower mean values for body weight gain and consequently body weight was observed from start of treatment onwards (3% lower body weight at Day 21 post-mating, compared to control), this was considered treatment related but not adverse.

Summarized data can be found in Attachment 2 (tables) and 1 (figures) under "Overall Remarks, Attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the low- and mid- dose group, no difference regarding food consumption was observed between control and treatment groups. In the high-dose group, mean food consumption was statistically significantly decreased during the first 6 days of treatment (in the intervals Days 9 - 12 and 12 - 15, up to 15% lower than control between Days 9 - 12 post-mating), followed by complete recovery to control levels up to the end of the treatment period. Over the whole treatment-period (Day 6 - 21 post-mating), food consumption in the high dose group was 8.85% decreased compared to the control group (statistically significant). These differences were considered treatment related but not adverse.

Summarized data can be found in Attachment 2 (tables) and 1 (figures) under "Overall Remarks, Attachments".

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

no effects observed

Description (incidence and severity):

No difference regarding water consumption was observed between control and treatment groups up to and including the highest dose level of 800 mg/kg bw/day.

Ophthalmological findings:

not examined

Description (incidence and severity):

not applicable

Haematological findings:

not examined

Description (incidence and severity):

not applicable

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not applicable

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

In the low- and mid- dose group, no difference in thyroid hormones was observed between control and treatment groups.
In the 800 mg/kg bw/day group, slightly increased serum levels of TSH were noted (1.4x of control). Even though, the difference did not reach statistical significance, a relation to treatment cannot be excluded. Levels of T3 and T4 were comparable to the control group. The differences were not considered adverse.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Thyroid weights and thyroid to body weight ratios of treated females were considered to be similar to those of control females.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Irregular surface of the non-glandular stomach was noted in 5/22 females at 800 mg/kg bw/day, and for 1/22 females at 300 mg/kg bw/day. As the test item is characterized as an irritant compound, a relation to treatment with the test item could not be excluded.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No difference regarding histopathology of the thyroid was observed between control and treatment groups.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions occurred.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No toxicologically relevant difference in pre- and post-implantation loss was observed between the control and treatment groups up to and including the highest dose level.
At 800 and 100 mg/kg bw/day, mean pre-implantation loss was slightly higher (10% and 8% vs 5% in the control group; not statistically significant), mainly attributed to 2 females each in the low- and high-dose group. However, as the treatment started on Day 6 post-mating (after implantation), the effect is not considered treatment-related.
In the high-dose group, mean post-implantation loss was slightly higher (7% vs 1% in the control group; not statistically significant). This was attributed mainly to one female with a relatively high number of early resorptions compared to the total number of implantations (n=6/10, respectively). As the post-implantation loss of all remaining high-dose females was either in the same range as for concurrent control females or only marginally higher and in absence of other developmental findings in this study, no toxicological significance was attached to this isolated finding.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No difference in total litter loss by resorption was observed between the control and treatment groups.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no late resorptions fetuses in this study.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead fetuses in this study.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No pre-mature deliveries occurred in this study

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No difference in pregnancy rate was observed between the control and treatment groups. At scheduled necropsy, 1/22 females of the control group, and 2/22 females of the 100 mg/kg bw/day group were non-gravid. The prematurely sacrificed female in the 1000 mg/kg bw/day group was non-gravid at early sacrifice.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Other effects:

no effects observed

Description (incidence and severity):

The number of females with viable litters was comparable between the control and treatment groups (21, 20, 22 and 21 in the control, 100, 300 and 800 mg/kg bw/day groups, respectively).

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed up to and including this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No differences in fetal body weight were observed between the control and treatment groups up.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences in the number of live fetuses were observed between the control and treatment groups.
In the high-dose group, mean litter size was slightly lower compared to the control group (-13%, not statistically significant) as a result of the lower number of implantation sites for 2 dams in this dose group. Therefore, the mean number of live fetuses at this high dose level was considered to reflect normal biological variation.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No treatment related difference in sex ratio was observed between the control and treatment groups up to and including the highest dose group.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No toxicologically relevant differences in the number of live fetuses were observed between the control and treatment groups up to and including the highest dose group.
In the high-dose group, mean litter size was slightly lower compared to the control group (-13%, not statistically significant) as a result of the lower number of implantation sites for 2 dams in this dose group. Therefore, the mean number of live fetuses at this high dose level was considered to reflect normal biological variation.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

No difference in anogenital distance was observed between the control and treatment groups.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant differences in the number of external malformations were observed between the control and treatment groups up to and including the highest dose group.
Two fetuses presented with external malformations. A single fetus at 100 mg/kg bw/day had exencephaly, and a single fetus at 800 mg/kg bw/day had a cleft palate and small upper jaw, but these were considered incidental.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant differences in the number of skeletal malformations were observed between control and treatment groups up to and including the highest dose group. 1 fetus of the 100 mg/kg bw/day group and 1 fetus of the 800 mg/kg bw/day group had multiple skeletal malformations consistent with their external findings (exencephaly and a cleft palate and a small upper jaw, respectively). The fetus of the 100 mg/kg bw/day group as well as one additional fetus of the 800 mg/kg bw/day group had sternoschisis. Due to the single occurrence in each group, these malformations were considered to be incidental findings. Additionally, the fetus from the 100 mg/kg bw/day group had limb malformations associated with the fibulas. Based on the single occurrence and the absence of a dose-related trend this finding was considered not treatment-related.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were observed in this study.
Visceral variations noted were supernumerary liver lobes (all groups) and a single discolored lung (several red spots; 300 mg/kg bw/day group). At the low incidence and in absence of a dose-related trend, these were considered not related to treatment with the test item.

Summarized data can be found in Attachment 2 under "Overall Remarks, Attachments".

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity and teratogenicity

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed up to and including the highest dose level tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The present study was conducted under GLP and according to OECD test guideline 414 (2018). Under the conditions of the study, administration of the test substance once daily by oral gavage for Days 6 - 20 post coitum was well tolerated in pregnant rats at levels up to 300 mg/kg bw/day. Based on mortality and lower body weight at 800 mg/kg bw/day, the NOAEL for maternal systemic toxicity was set at 300 mg/kg bw/day. No test item-related adverse findings were observed regarding developmental toxicity and teratogenicity. Therefore, the NOAELs for developmental toxicity and teratogenicity were set at 800 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus 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 alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) as well as several member substances of the Alcohol Ethoxylates (AE) category.

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

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) was tested in Wistar Han rats in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test according to OECD guideline 422 under GLP conditions (Innospec, 2020). 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).

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92, 86, 87 and 82% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. It should be noted that one female had 2 pups compared to 18 uterine implantation sites, which attributed to the slightly lower post-implantation survival index of the 1000 mg/kg bw/day group. As this occurred for one animal only, this was considered not toxicologically relevant. The total number of offspring born compared to the total number of uterine implantations was consequently considered not adversely affected. Litter size was not affected by treatment: Live litter sizes were 10.2, 10.7, 11.9 and 9.7 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The live birth indices were 97, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Three pups of the control group (all in one litter) were found dead at first litter check. No toxicological relevance was attributed to these deaths since it occurred in the control group. 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 100, 100, 98 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Two pups at 300 mg/kg bw/day (both in the same litter) were found dead or missing on PND 2 or 4. The 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, 98, 98 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup at 100 mg/kg bw/day, was euthanised in extremis on PND 6 as this animal was dehydrated, thin and weak. As this was the only pup of the respective female, this was a total litter loss. One pup at 300 mg/kg bw/day was found dead on PND 5. 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. No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of the clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant. Lower body weights of pups at 1000 mg/kg/day were noted from PND 1 onwards (up to 0.85x and 0.87x of control in male and female pups, respectively; not always statistically significant). Other statistically significant changes in body weights of the pups were considered to be unrelated to treatment as these occurred in absence of a dose-related trend. Sex ratio and anogenital distance (absolute and normalized for body weight) in male and female pups were considered not to be affected by treatment with the test item. Treatment up to 1000 mg/kg bw/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. Finally, no macroscopic findings were noted. 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. No indication of a teratogenic effect was observed at any dose level.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of 300 mg/kg bw/day for developmental toxicity was determined. It is important to note that the reduced pup weights observed in this study occurred at dose levels associated with maternal systemic toxicity. This included clinical signs for females during the entire study period. It is reasonable to assume that these clinical signs were related to the observed developmental toxicity. The reduced pup weights observed in the high-dose group (1000 mg/kg bw/day) can therefore be regarded as a secondary effect to the systemic maternal toxicity. This conclusion is further supported by the fact that no developmental toxicity was observed at dose levels which were non-toxic to the dams.

Alcohols, C9-11, branched and linear, ethoxylated (CAS 160901-09-7, EC No. 500-446-0) was administered to female Wistar (Crl: WI(Han) rats in a Prenatal developmental toxicity study according to OECD guideline 414 under GLP conditions (Innospec, 2022b). Groups of 22 dams were administered doses of 100, 300 and 800 mg/kg bw/day by oral gavage, 7 days a week on gestation day 6 - 20. The control group was treated according to the same protocol and received the vehicle (corn oil) only. All the females were time-mated and the day of mating was gestation day 0 (Day 0 post-coitum). The dams were sacrificed on gestation day 21.

The following parameters were recorded in the dams: mortality/moribundity, clinical signs, body weight and food consumption, water consumption (monitored by visual inspection), thyroid weight, measurement of thyroid hormones (T3, T4 and TSH), macroscopic examination, gravid uterus weight, number of corpora lutea, number of implantations, number of early and late resorptions, and number of live and dead fetuses. The following parameters were recorded in the foetuses: litter size, body weight, sex, anogenital distance of all live pups, external examination, soft tissue examination, head examination and skeletal examination.

A female in the high-dose group was sacrificed in extremis on gestation day 12. The female had laboured breathing, had a weak appearance, was lying in a recumbent position and was cold to the touch. Slight body weight loss (3%) was noted Day 6 - 9 post-mating, followed by no recovery thereafter. In addition, the food consumption was lower Day 6 - 12 post-mating than during the acclimatization period. At necropsy, no macroscopic alterations were observed and the female was found to be non-gravid. No definite cause of moribundity could be determined. However, based on the available data a possible relation to treatment with the test item could not be excluded.

Salivation was seen following dosing on several occasions during gestation day 12 - 20 in 3/22 mid-dose and 11/22 high-dose females. The salivation was not considered toxicologically relevant and was considered a physiological response following treatment rather than a sign of systemic toxicity. Abnormal breathing sounds were observed in0/22, 2/22, 4/22 and 9/22 females in the control, low- mid- and high-dose group, respectively, throughout the dosing period. Incidental cases of erected fur in up to 3/22 females in the mid-dose and high-dose groups was noted. These effects were considered treatment-related but not adverse.

In the high-dose group, the terminal body weight was 3% lower than control values (not significant). The mean gravid uterus weight was 14% lower than control (not statistically significant). This was mainly due to 3 individual females with small litters (2 - 4 fetuses). As these differences were slight and not statistically significant, they were not considered toxicologically relevant.

The food consumption was reduced during the complete dosing period in high-dose females, compared with the control. During gestation day 6 – 21 the mean overall food intake was -9% compared to the control. The reduced food consumption is not considered to be toxicologically relevant, as it was limited and did not affect the body weight.

Slightly increased serum levels of TSH (1.4 x) were noted in high-dose females, but did not reach statistical significance. As the mean and most individual values remained within the historical control data range, and in the absence of any macroscopic observations of the thyroid gland, this slight increase was considered not to be adverse.

An irregular surface of the non-glandular stomach was noted in 1/22 mid-dose and 5/22 high-dose females. As the test substances has irritating properties, the effect is likely to be caused by the treatment.

In high-dose females the mean post-implantation loss was slightly higher (7% vs 1% in the control group; not statistically significant). This was attributed mainly to one female with a relatively high number of early resorptions compared to the total number of implantations (n=6/10). As the post-implantation loss of all the remaining high-dose females was in the same range as for the control females or only marginally higher and in absence of other relevant developmental findings, this is considered to be a non-adverse finding.

No treatment-related effects were noted on thyroid weight and microscopy, litter loss, number of corpora lutea, number of pre-implantations, number of early and late resorptions, and number of live and dead fetuses.

The maternal NOAEL systemic was set at 300 mg/kg bw/day, based on the unscheduled sacrifice of one female in the high-dose group.

The mean litter size in the high-dose group was slightly lower (13%) than in the control (not statistically significant). As this was the result of the lower number of implantation sites for 2 females, the mean number of live fetuses at this high dose level was considered to reflect the normal biological variation.

External malformations were observed in one fetus in the low-dose group and in one fetus in the high-dose group. There were no treatment-related visceral malformations and a few variations in all the groups. Three fetuses had a skeletal malformation; one in the low-dose group and two in the high-dose group. All these cases were considered incidental findings due to the single or low occurrence. No treatment-related effects were noted on litter size, offspring body weight, sex ratio and anogenital distance.

The NOAEL developmental is 800 mg/kg bw/day and the NOAEL teratogenicity is 800 mg/kg bw/day.

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 alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) is consistent with the overall toxicity to reproduction data for AE substances.

The following NOAELs were set:

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 800 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 800 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 alcohols, C9-11, branched and linear, ethoxylated (CAS No. 160901-09-7, EC No. 500-446-0) and with other members of the Alcohol Ethoxylates (AE) category do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.