Alcohols, C12-13, 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) ≥ 1000 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, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 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

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Dec 2019 - 26 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

GLP compliance:

yes (incl. QA statement)

Remarks:

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

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: Males 10-11 weeks, females 13-14 weeks of age
- Weight at study initiation: Males 278 - 353 g, females 198 - 239 g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 45 - 53
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

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.

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.

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. 20218719).

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 males and 10 females for each treatment group and control group

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of Alcohols, C12-13, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item (Test Facility Reference No. 20218720).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted once 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, prostate weight, seminal vesicle weight

Litter observations:

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

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

GROSS EXAMINATION OF DEAD PUPS: Pups found dead were examined for external and internal abnormalities.
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.

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.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups were euthanized on PND 14-16, except for the two pups per litter selected for blood collection which were euthanized by an intraperitoneal injection of sodium pentobarbital.

The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

Pups that died before scheduled termination were examined externally and sexed (both externally and internally).

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

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.

HISTOPATHOLOGY / ORGAN WEIGTHS
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 reproductive indices calculations were performed. Group mean values of precoital time and duration of gestation 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 1x 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

Offspring viability indices:

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):

Test item-related clinical signs of toxicity were observed in treated animals during daily detailed clinical observations.

Group 2 (100 mg/kg bw/day): In one male red staining of the nose was recorded on six consecutive days during the mating period, after which it recovered. No clinical signs of toxicity were observed in females at 100 mg/kg bw/day.

Group 3 (300 mg/kg bw/day): In three males and two females clinical signs of toxicity were observed on 1-3 days of treatment, at the beginning of Week 2 or during post-coitum, in one male red staining of the mouth was recorded on Days 8-9 of treatment. No other clinical signs were recorded for this animal. Two males and one female showed slight breathing rales on Day 10 of treatment. In the same female ventro-lateral recumbency and pon Days 9-10 post-coitum. One female had breathing rales on three consecutive days during Week 2 of lactation.

Group 4 (1000 mg/kg bw/day): Males and females at 1000 mg/kg bw/day showed clinical signs of toxicity at the end of Week 1/beginning of Week 2 of treatment, which lasted for several days and then recovered. In females, clinicals signs were also recorded during lactation. In three males at 1000 mg/kg bw/day flat and/or hunched posture, lethargy, piloerection and/or ptosis of the eyes were recorded on Days 7-8 of treatment. Red staining of the mouth was recorded in one male on Day 8 of treatment. Breathing rales were observed in six males on one or two days during Week 2 of treatment, and in one male on two days in Week 4 of treatment. In all females at 1000 mg/kg bw/day flat and hunched posture, breathing rales (slight to moderate) and piloerection were observed on two or more occasions from Day 6 up to Day 13 of treatment and during lactation. Slight ptosis of the eyes was recorded on two or more occasions from Day 9 up to Day 13 of treatment. Salivation seen after dosing among animals of the 100, 300 and 1000 mg/kg bw/day dose groups starting at Week 1 or 2 of the treatment period was considered not toxicologically relevant, taking into account the nature and 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 incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed in females up to 1000 mg/kg bw/day.

In males receiving 300 and 1000 mg/kg bw/day test material, body weight gain was decreased from Day 8 of treatment onwards (statistically significant on some occasions for animals receiving 1000 mg/kg bw/day only). Decreased body weight gain resulted in body weights up to 0.95x (300 mg/kg bw/day) and 0.94x (1000 mg/kg bw/day) of control (not statistically significant). These differences were considered to be test item-related.

Statistically significant changes in body weight gain in females at 100 and 300 mg/kg bw/day during lactation were considered to be unrelated to treatment since all values were within the normal range and no dose-related trend was apparent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment up to 1000 mg/kg bw/day.
Food consumption in males and females at 1000 mg/kg bw/day was decreased in Week 1 of treatment (up to 0.87x of control for relative food consumption, not statistically significant). As this observation was transient, it 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):

Decreased mean corpuscular volume (MCV) was seen in males and females at 1000 mg/kg bw/day (0.96x of control, not statistically significant). Mean values for males were just below the historical control range; for females mean values remained within the normal range. Considering the magnitude of change, this difference was considered not toxicologically relevant.

Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend. Increased reticulocyte concentrations in females starting at 100 mg/kg bw/day were considered the result of relatively low control values and not related to treatment with the test item.

Decreased activated partial thromboplastin time (APTT) in females starting at 100 mg/kg bw/day were considered the result of relatively high control values and not related to treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urea levels were seen in males at 1000 mg/kg bw/day (1.49x of control). Mean values were outside the historical control range. This change was considered test item-related.

Increased creatinine concentrations were seen in males and decreased creatinine concentrations in females at 1000 mg/kg bw/day (1.09x and 0.91x of control, respectively). Mean values were outside the historical control range. This change was considered test item-related.

Increased bile acids in males and females was seen starting at 100 mg/kg bw/day (not statistically significant, up to 1.66x of control) but were considered not toxicologically relevant as mean values remained within the historical control range, no dose-response was apparent (males) or mean differences were the result of high variation (females).

Statistically significantly decreased calcium concentrations in males at 300 mg/kg bw/day were 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 considered unaffected by treatment with the test item up to 1000 mg/kg bw/day. At 1000 mg/kg bw/day, serum T4 levels were slightly decreased when compared to the concurrent control group (0.82x of control). However, as no statistical significance was reached, the magnitude of change was minimal, and all values were within normal limits of which the concurrent control was at the upper end, this difference was considered not related to treatment with the test item.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment in males up to 1000 mg/kg bw/day, and in females up to 300 mg/kg bw/day. Hearing ability, pupillary reflex, static righting reflex and grip strength of the hind leg were normal in all examined animals up to 1000 mg/kg bw/day.
In females at 1000 mg/kg bw/day, grip strength of the fore leg, total movements and ambulations were decreased (0.79x, 0.59x and 0.68x of control, respectively, statistically significant for grip strength only). Mean values remained within the historical control range. These changes were considered test item-related.

In the absence of a dose-related trend, statistically significantly increased ambulations in males at 300 mg/kg bw/day were considered unrelated to treatment. 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 at 1000 mg/kg/day in the liver of males and adrenal gland of females. These findings are presented in Table 2.

Liver (males): Centrilobular hepatocellular hypertrophy was noted in all males at 1000 mg/kg/day at minimal to slight degree.

Adrenals gland (females): Cortical hypertrophy of the zona fasciculata was recorded in 2/5 females at 1000 mg/kg/day at minimal degree.

In addition, in the heart of a single male at 1000 mg/kg/day multifocal necrosis of myofibres (left ventricle) at slight degree was recorded for which a relation to treatment could not be ruled out.
The remainder of the recorded microscopic findings (including hypertrophy of follicular cells in the thyroid gland) were within the range of background pathology encountered in rats of this age and strain subjected to a combined 28-Day oral toxicity study. 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. Most females had regular cycles of 4 to 5 days. Extended di-estrous occurred in one female at 1000 mg/kg bw/day, due to which the regularity of the estrous cycle could not be determined. Given the incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was considered not to be affected by treatment with the test item. All females showed evidence of mating. Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days. Number of implantation sites was considered not to be affected by treatment with the test item. Mean number of implantation sites was 11.2, 12.8, 12.2 and 11.2 in the control, 100, 300 and 1000 mg/kg/day groups, respectively. Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 90% for the control, 100 and 300 mg/kg bw/day groups and 100% for the 1000 mg/kg bw/day group. One female of the control, 100 and 300 mg/kg bw/day groups each were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

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:

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. The nature and incidence of 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):

One pup of the control group was found missing on PND 5. In addition, one pup at 100 mg/kg bw/day was found missing on PND 16. 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, body weights of male and female pups were decreased from PND 1 onwards (statistically significant from Day 7 onwards). Body weights progressively decreased from 0.89x of control on Day 1 up to 0.80x of control on PND 13. This was considered test item-related. At 100 and 300 mg/kg bw/day male and females pup body weights were slightly lower than control as well, but not progressive (up to 0.94x of control). As differences were minimal these were considered not adverse.

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.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

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. The statistically significantly lower mean anogenital distance (normalized for body weight) of female pups at 300 mg/kg bw/day occurred without a dose related-trend and was not considered treatment-related.

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 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, 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

Remarks:

development

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

Key result

Reproductive effects observed:

no

Table 1:  Mean Percent Liver and Adrenal Glands Weight Differences from Control Groups

	Males					Females
Dose level (mg/kg/day):	100		300	1000		100	300	1000
LIVER
Absolute	7		-4	29**		12	13	34**
Relative to body weight	5		3	35**		9	14**	34**
ADRENAL GLANDS
Absolute	-		-	-		21	9	32*
Relative to body weight	-		-	-		17	8	33*

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

Table 2: Summary Test Item-Related Microscopic Findings

Dose level (mg/kg/day):	0	100	300	1000
LIVER (males)^a	5	5	5	5
Hypertrophy hepatocellular              Centrilobular
Minimal	-	-	-	3
Slight	-	-	-	2
ADRENAL GLANDS (females)^a	5	5	5	5
Hypertrophy cortical            zona fasciculata
Minimal	-	-	-	2

^a = Number of tissues examined from each group.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Please refer to the category justification provided in the category object.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

For a detailed assessment of the reproductive / developmental toxicity of the Alcohol Ethoxylates (AE) category, please refer to the category justification attached to the category object.

Conclusions:

Applying read-across based on grouping of substances (category approach), no toxicologically relevant effects in relation to reproductive / developmental toxicity and a NOAEL for reproductive toxicity ≥ 1000 mg/kg bw/day are predicted for the registered substance.

Executive summary:

The available data on reproductive / developmental toxicity in the 'linear' subgroup of the Alcohol Ethoxylates (AE) category indicate no toxicologically relevant effects for the registered substance. As explained in the category justification, the differences in molecular structure and composition between the registered substance and the members of the AE category are unlikely to lead to differences with respect to reproductive / developmental toxicity.

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 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, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 

The reproductive toxicity of alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 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 (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 50-56 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver or had a total litter loss were treated for 40-43 days. The following parameters and endpoints relevant for reproductive toxicity were evaluated: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care. In addition, a number of developmental parameters as well as endpoints indicative of repeated dose toxicity were investigated. The details of the assessment of developmental toxicity are summarised below under 'Additional information' in the section for effects on developmental toxicity and the repeated dose toxicity details are provided in IUCLID section 7.5.1.

Most females had regular cycles of 4 to 5 days. Extended di-estrous occurred in one high-dose female (1000 mg/kg bw/day), due to which the regularity of the estrous cycle could not be determined. Given the incidental nature and absence of an apparent correlation to pregnancy status, the finding did not indicate a relation with treatment. Mating index was considered not to be affected by treatment with the test item. All females showed evidence of mating. Precoital time was not affected by treatment as all females showed evidence of mating within 4 days. The mean number of implantation sites was 11.2, 12.8, 12.2 and 11.2 in the control, 100, 300 and 1000 mg/kg/day groups, respectively. Thus, the number of implantation sites was considered not to be affected. The fertility indices were 90% for the control, 100 and 300 mg/kg bw/day groups and 100% for the 1000 mg/kg bw/day group. One female of the control, 100 and 300 mg/kg bw/day groups each were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment. No abnormalities were seen in the reproductive organs of the animals, which could account for their lack of offspring. 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. Gestation index and duration of gestation were not affected by treatment. The gestation indices were 100% for the control, 100 and 300 mg/kg bw/day groups and 90% for the 1000 mg/kg bw/day group. The failed pregnancy of one female, which had 2 implantation sites only, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of ≥ 1000 mg/kg bw/day for reproductive toxicity 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 alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 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) = 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) = 1000 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 1000 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 400 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 400 mg/kg bw/day

 

Read-across based on grouping of substances (category approach) considering all the available data on developmental toxicity in the AE category, in a Weight-of-Evidence approach.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Please refer to the category justification provided in the category object.

Species:

rat

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: clinical signs of systemic toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weights as a secondary effect to systemic maternal toxicity

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: reduced pup weights as a secondary effect to systemic maternal toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

For a detailed assessment of the developmental toxicity of the Alcohol Ethoxylates (AE) category, please refer to the category justification attached to the category object.

Conclusions:

Applying read-across based on grouping of substances (category approach), reduced pup weights and a NOAEL for developmental toxicity of 300 mg/kg bw/day are predicted for the registered substance. Reduced pup weights are considered to be a secondary effect to systemic maternal toxicity.

Executive summary:

The available data on developmental toxicity in the 'branched & linear' subgroup of the Alcohol Ethoxylates (AE) category indicate reduced pup weights for the registered substance. Reduced pup weights are considered to be a secondary effect to systemic maternal toxicity. As explained in the category justification, the differences in molecular structure and composition between the registered substance and the members of the 'branched & linear' subgroup of the AE category are unlikely to lead to differences with respect to developmental toxicity.

Pursuant to compliance check final decision for several branched & linear AE substances, prenatal developmental toxicity studies in a first (rat) and/or a second (rabbit) species according to OECD guideline 414 are currently ongoing. The NOAEL for developmental toxicity established for alcohols, C12-13, branched and linear, ethoxylated, < 2.5 EO (CAS No. 160901-19-9, EC No. 500-457-0) will be re-evaluated once the results of the OECD 414 studies conducted with other branched & linear AE substances are available.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Please refer to the category justification provided in the category object.

Species:

rabbit

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: clinical signs of systemic toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced pup weights as a secondary effect to systemic maternal toxicity

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: reduced pup weights as a secondary effect to systemic maternal toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

For a detailed assessment of the developmental toxicity of the Alcohol Ethoxylates (AE) category, please refer to the category justification attached to the category object.

Conclusions:

Applying read-across based on grouping of substances (category approach), reduced pup weights and a NOAEL for developmental toxicity of 300 mg/kg bw/day are predicted for the registered substance. Reduced pup weights are considered to be a secondary effect to systemic maternal toxicity.

Executive summary:

The available data on developmental toxicity in the 'branched & linear' subgroup of the Alcohol Ethoxylates (AE) category indicate reduced pup weights for the registered substance. Reduced pup weights are considered to be a secondary effect to systemic maternal toxicity. As explained in the category justification, the differences in molecular structure and composition between the registered substance and the members of the 'branched & linear' subgroup of the AE category are unlikely to lead to differences with respect to developmental toxicity.

Pursuant to compliance check final decision for several branched & linear AE substances, prenatal developmental toxicity studies in a first (rat) and/or a second (rabbit) species according to OECD guideline 414 are currently ongoing. The NOAEL for developmental toxicity established for alcohols, C12-13, branched and linear, ethoxylated, < 2.5 EO (CAS No. 160901-19-9, EC No. 500-457-0) will be re-evaluated once the results of the OECD 414 studies conducted with other branched & linear AE substances are available.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

400 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies from various source substances in the Alcohol Ethoxylates (AE) category with similar structures and intrinsic properties. Read-across is justified based on common toxicokinetic behaviour and consistent trends in environmental fate, ecotoxicological and toxicological properties of the category member substances. The data pool of the AE category is thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.7, in accordance with Annex XI, Section 1.5, 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

No data on prenatal developmental toxicity are available for alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0), while some information on developmental toxicity is available from the screening study (OECD 422). In order to assess the potential for developmental toxicity, the screening study and studies in the database of the Alcohol Ethoxylates (AE) category are considered in a read-across approach.

Study with alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0)

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 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 (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 with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 85, 92, 90 and 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Litter sizes at first litter check were 9.6, 11.7, 11.0 and 10.9 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Control values were slightly low. Litter size was therefore not affected by the treatment. The number of live offspring on Day 1 after littering compared to the total number of offspring born was also not affected. The live birth indices were 100, 99, 100 and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup at 100 mg/kg bw/day and two pups at 1000 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 not affected by the treatment. Viability indices were 100% for the control, 100 and 300 mg/kg bw/day groups and 99% for the 1000 mg/kg bw/day group. One pup at 1000 mg/kg bw/day was found missing on PND 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to this missing pup 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 not affected by the treatment. The lactation indices were 98% for the control group and 100% for the 100, 300 and 1000 mg/kg bw/day groups. One pup of the control group was found missing on PND 5. In addition, one pup at 100 mg/kg bw/day was found missing on PND 16. 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. 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, and were therefore considered not to be toxicologically relevant. At 1000 mg/kg bw/day, body weights of male and female pups were decreased from PND 1 onwards (statistically significant from Day 7 onwards). Body weights progressively decreased from 0.89x of control on Day 1 up to 0.80x of control on PND 13. This was considered test item-related. At 100 and 300 mg/kg bw/day male and females pup body weights were slightly lower than control as well, but not progressive (up to 0.94x of control). As differences were minimal these were considered not adverse. 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. The statistically significantly lower mean anogenital distance (normalised for body weight) of female pups at 300 mg/kg bw/day occurred without a dose related-trend and was not considered treatment-related. 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. No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment. Hence, 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 in males and females comprising of red staining of the mouth and lethargy (males only), breathing rales (slight to moderate), flat and/or hunched posture, piloerection and ptosis of the eyes. 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.

Studies in the AE category

Studies investigating 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	300	≥ 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

 

Evaluation of developmental toxicity as observed in available studies

The database for reproductive and developmental toxicity consists of the eight combined repeated dose toxicity studies with the reproduction / developmental toxicity screening test (OECD guideline 422), which are discussed under “Effects on fertility” in section 7.8 and in the repeated dose toxicity section 7.5. In addition, four prenatal developmental toxicity studies (OECD guideline 414) in rats and two prenatal developmental toxicity studies in rabbits (two rat studies and one rabbit study in each of the ‘linear’ and ‘mixed branched & linear’ subgroups, respectively) are available. A third prenatal developmental toxicity study in rabbits is currently ongoing. It has been delayed due to the high workload of the test facility. As soon as reliable results are available, the hazard assessment with respect to developmental toxicity in rabbits will be updated.

Certain effects were noted in several of the studies, although the effects were not always considered to be adverse or toxicologically relevant (please refer to the endpoint summary 7.5 Repeated dose toxicity for more details). As can be expected for surfactants with known irritating properties, some effects caused by irritation at the site of contact (fore-stomach) were observed in the studies in the rat. The effects observed in the prenatal developmental toxicity studies are summarised and discussed below.

 

Prenatal developmental toxicity studies in rats

The prenatal developmental toxicity studies were performed according to OECD guideline 414. Groups of 22 dams were administered the test substance by oral gavage, 7 days a week on gestation day 6 – 20. The dose levels applied were either the guideline-recommended top dose of 1000 mg/kg bw/day or were established in appropriate dose range-finding pre-studies that identified top doses inducing significant toxicity without imposing unnecessary suffering on the laboratory animals. 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 hormone levels (T3, T4 and TSH), macroscopic examination, microscopic examination of the thyroid gland, 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: body weight, sex, anogenital distance, external examination, soft tissue examination and skeletal examination for malformations and variations.

In the study with alcohols, C12-14, ethoxylated it was suspected that subgroups 1 and 2 may have been switched after mating. This would indicate that animals of subgroup 1 were dosed on gestation day 5 - 19 and sacrificed on gestation day 20, and animals of subgroup 2 were dosed on gestation day 7 - 21 and sacrificed on gestation day 22. Subgroups 3 and 4 were dosed according to schedule. A full evaluation of the data was still possible, as subgroups 3 and 4 were dosed according to schedule and sufficient data was available, all dose groups were evenly divided over the different subgroups, data of applicable parameters were evaluated separately for the different subgroups, if deemed necessary, and possible test item-related trends were consistent between the different subgroups. Furthermore, for the majority of animals, the critical period of organogenesis was completely included in the dosing period the suspected slight shift in dosing period for subgroups 1 and 2 (i.e., one day earlier and one day later, respectively) had no impact on the overall conclusion. It was therefore concluded that the overall integrity of the study and the interpretation of the study results and conclusions was not compromised.

 

Maternal effects

Maternal effects observed in the studies were mostly related to clinical signs and body weight development. Clinical signs included salivation, piloerection, hunched posture and abnormal breathing sounds and were considered a physiological response to treatment rather than signs of systemic toxicity. Terminal body weights and body weight gain were lower in high-dose females than in the control in all four OECD 414 studies in the rat. In the study with alcohols, C12-14, ethoxylated this resulted in terminal body weights of 7 - 11% lower than controls. The mean overall food intake was reduced by 11 - 24% compared with controls. While effects on body weight development were considered adverse for alcohols, C12-14, ethoxylated, they were not considered adverse for the other three OECD 414 studies in rats due to the limited difference between the treated and control animals. The body weight gain corrected for gravid uterus was slightly reduced compared with control in all four studies, but not considered a toxicologically relevant effect as the difference was limited and not significant.

A reduction in serum levels of total T3 and T4 was noted in the study with alcohols, C16-18 and C18-unsatd., ethoxylated, but the mean value of both parameters remained within the historical control data. Reduced levels of T3 and increased levels of TSH were also noted in the studies with alcohols, C12-14, ethoxylated and alcohols, C12-15, branched and linear, ethoxylated. As there were no correlated histopathological changes and the values remained within the range of the historical control data, the changes in thyroid hormones were not considered to be toxicologically relevant.

In the study with alcohols, C9-11, branched and linear, ethoxylated, a single (non-pregnant) female of the high-dose group (800 mg/kg bw/day) was sacrificed in extremis on gestation day 12, as it was in poor health with laboured breathing, lateral recumbent position, a weak appearance and decreased activity, cold to the touch and slight salivation. The days before its death, slight body weight loss was noted for gestation day 6 - 9, with no recovery thereafter. In addition, the food consumption was lower during gestation day 6 - 12. Although no definite cause of the poor health could be determined at necropsy (i.e. no macroscopic abnormalities), a possible relation to treatment with the test substance could not be excluded. In the study with alcohols, C9-11, branched and linear, ethoxylated the macroscopic evaluation showed an irregular surface in the forestomach of 1/22 females in the mid-dose group and 5/22 females of the high-dose group, respectively. No microscopic evaluation was performed. However, this finding is in agreement with the observations in the subacute and subchronic repeated dose toxicity studies with the substance, and it is classified for its eye irritation potential. It is likely that the effects is caused by a local irritant effect.

No treatment-related and/or toxicologically relevant changes were noted for any of the other maternal systemic and reproductive parameters recorded in the studies (thyroid gland weights, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, pre- and post-implantation sites, and pre- and post-implantation loss).

 

Effects on offspring

The mean fetal body weights (male, female and combined) were reduced, compared with control, in the studies with alcohols, C12-14, ethoxylated and alcohols, C16-18 and C18-unsatd., ethoxylated. For alcohols, C12-14, ethoxylated the effect was considered adverse as the mean values were below or at the lower end of the historical control range, while for alcohols, C16-18 and C18-unsatd., ethoxylated the effect was not considered to be adverse as the difference in mean body weight was around 3% for all treatment groups.

In the study with alcohols, C12-14, ethoxylated effects relating to offspring development were found. These effects, noted in the high-dose group (1000 mg/kg bw/day), were all skeletal variations: signs of delays of ossification (unossified metacarpals, sternebrae and hyoid body, incompletely ossified sternebrae, skull bones (e.g., supraoccipital, parietal and frontal), vertebra (cervical and sacral) and pelvic girdles (ischium)). Incompletely ossified skull bones also occurred across the control and low-dose group (100 mg/kg bw/day). None of the findings was considered adverse as the effects are defined as a variation. The degree of ossification was correlated with the body weight of the respective foetuses and the body weights varied by subgroup, due to the inadvertent switch of two subgroups. A few cases of external, visceral and skeletal malformations and -variations were recorded in the remaining three studies in the rat, included cleft palate, a small upper jaw, exencephaly, dilated brain ventricles and bent humeri. These cases were all considered to be incidental due to the low number or were observed in all groups including control in similar numbers.

No toxicologically relevant changes were observed in any of the remaining developmental parameters investigated in the studies (litter size, sex ratio, anogenital distance and placental weights of live foetuses).

Prenatal developmental toxicity studies in rabbits

The prenatal developmental toxicity studies were performed according to OECD guideline 414. Groups of 22 dams were administered the test substance by oral gavage, 7 days a week on gestation day 7 – 28. The dose levels applied were established in appropriate dose range-finding pre-studies that identified top doses inducing significant toxicity without imposing unnecessary suffering on the laboratory animals. The control group was treated according to the same protocol and received the vehicle (propylene glycol) 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 29.The following parameters were recorded in the dams: mortality/moribundity, clinical signs, body weight and food consumption, water consumption (monitored by visual inspection), 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: body weight, sex, external examination, soft tissue examination and skeletal examination for malformations and variations.

 

Behavioural episodes observed in dams

In both of the available prenatal developmental toxicity studies in rabbits incidences during which individual dams showed severe clinical signs were noted. In the study with alcohols, C12-14, ethoxylated, the episodes occurred on several (consecutive) days of treatment from gestation day 15 onwards in 5/22 animals of the high-dose group (200 mg/kg bw/day). The episodes consisted of the following clinical signs: increased respiratory rate, shallow breathing, uncoordinated movements, lying on one side, head tilt, prostrate position, decreased activity and/or extensor rigidity of both hind limbs, prior to and/or during the first post-dose observation. In addition, uncoordinated movement and/or lying on the side was noted in 18/22 and 16/22 animals, respectively, in the high-dose group. The episodes were not observed in the low- (30 mg/kg bw/day) and mid-dose group (80 mg/kg bw/day).

In the high-dose group (400 mg/kg bw/day) of the study with alcohols, C12-15, branched and linear, ethoxylated, 2/19 females similar incidences were noted on gestation day 20 and 22, 2-4 h post-dose. A combination of the following clinical signs was observed: uncoordinated movements with extensor rigidity of both hind limbs, rolling and/or shallow breathing, decreased activity, limited use of both hind limbs, lameness of both hind limbs, abnormal gait, lying on the side, difficulty rising and/or abnormal respiratory rate. Extensor rigidity of both hind limbs, lying on the side, prostrate position, erected fur and/or hunched posture was also noted in these two females as well as in three additional females of this dose group on individual days between gestation day 23 and 28. Moreover, one female in the mid-dose group (250 mg/kg bw/day) showed extensor rigidity of both hind limbs, lying on the side, prostrate position, erected fur and/or hunched posture on individual days between gestation day 23 and 28, mostly 2-4 h post-dose.

The behavioural episodes or individual clinical signs associated with them, e.g. uncoordinated movement and extensor rigidity of both hind limbs, started around gestation day 15, about 7-10 days after the treatment with the test substances started. Only a minority of the females were subject to the episodes. Clinical signs observed in other females included decreased activity, hunched posture, erected fur and prostrate position. These clinical signs are probably a consequence of a disturbed well-being of the pregnant rabbits as a result the treatment. They do not appear to be associated with the episodes.

The cause of the episodes is unknown. It is notable that they started around 7-10 days after initiation of the dosing and that only a fraction of the test animals - even in the high-dose groups - was subject to all the clinical signs associated with the ‘episodes’. Certain clinical signs characteristic for the episodes were also observed in other females. It is well-known that rabbits - and especially pregnant rabbits - are more sensitive than other laboratory animals in general and may consequently show more (severe) adverse effects of treatment with a xenobiotic test material. However, no comparable observations were found in the toxicological literature and none of the toxicological and veterinary experts involved in (evaluation of) the studies was aware of similar effects in rabbits during studies. There were no indications of similar or comparable effects found in the prenatal developmental toxicity studies in rats.

One possible explanation of the episodes that was considered is that they indicate a rabbit-specific metabolisation of AE substances leading to metabolites responsible for the strong effects observed in rabbits but not in rats and other species. Since the ‘episodes’ were reported to occur approx. 1 to 4 h after dosing, it is possible that metabolisation of the substances’ constituents is needed, i.e. that certain metabolites are responsible for the severe clinical signs. In order to evaluate this assumption, the results of the comparative in vitro metabolisation investigation reported in section 7.3 were evaluated in the context of the observed clinical signs. The study did not reveal substantial species differences with respect to the metabolisation of AE substances. Different metabolisation rates and half-lives were observed as can be expected for different species. However, the metabolites identified after incubation with human, rat and hamster liver S9 fractions and hepatocytes did not differ qualitatively. Although the amount of certain metabolites varied between the species, no differences in metabolic pathways were identified. All three investigated species (human, rat and hamster) produced the same metabolite spectrum. For a detailed discussion of the comparative in vitro metabolism investigation performed with selected AE test substances and of the ‘common underlying mechanism’, please refer to section 7. The findings of the in vitro metabolism investigation are a clear indication that there are no species differences in relation to the metabolism of AE substances. This result is also supported by studies reviewed in the HERA report (refer to section 7.1). There are no indications that AE substances are metabolised in a different way in rabbits when compared to humans, rats and hamsters. Based on all available information, it is reasonable to assume that the episodes observed in the rabbit studies - but not in any of the available studies in rats - are a consequence of the generally higher sensitivity of pregnant rabbits to treatment under similar conditions. In addition to the generally higher sensitivity of rabbits under similar conditions, it should be noted that the studies in rats were all performed using corn oil as vehicle whereas propylene glycol had to be used in the rabbit studies. While corn oil is known to mitigate effects of local irritation, propylene glycol cannot reduce local irritation effects. The treated rabbits may consequently be more affected by the gavage exposure to an irritant substance. The difference between using corn oil and propylene glycol as the vehicle in combination with the generally higher susceptibility of pregnant rabbits, compared with rats, to treatment under similar conditions may contribute to the ‘episodes’. Therefore, the behavioural episodes are not considered to be an indication of a rabbit-specific systemic toxicity. Since an additional prenatal developmental toxicity study in rabbits is currently ongoing with alcohols, C16-18 and C18-unsatd., ethoxylated, a final evaluation of the effects observed in pregnant rabbits can only be made when the results of this study will be available.

 

Other maternal effects

Up to 3 females died or were sacrificed in extremis prior to scheduled sacrifice in all the control and treatment groups, in both studies. This was partly due to the behavioural episodes described above, partly due to animals that were in poor health as they were not eating enough, partly caused by gavage error, and partly due to early delivery. In the study with alcohols, C12-14, ethoxylated, one case in the high-dose group was considered adverse as this animals had severe behavioural episodes. In the study with alcohols, C12-15, branched and linear, ethoxylated, one case in the mid-dose group and three in the high-dose group were considered adverse due to body weight loss and clinical signs. The mean overall body weight gain was reduced by 7-11% in all treatment groups in the study with alcohols, C12-14, ethoxylated. The changes were limited and not statistically significant, and therefore not considered to be toxicologically relevant. In the study with alcohols, C12-15, branched and linear, ethoxylated the overall body weight gain was reduced by 35%, compared with control (not statistically significant). This was a toxicologically relevant effect due to the large change. The reduction in food consumption was considered toxicologically relevant only in the high-dose group of the study with alcohols, C12-15, branched and linear, ethoxylated as it was 18% lower than for control. 

Terminal body weights and body weight gain were lower in high-dose females than in the control in all four OECD 414 studies in the rat. In the study with alcohols, C12-14, ethoxylated this resulted in terminal body weights of 7 - 11% lower than controls. The mean overall food intake was reduced by 11 - 24% compared with controls. While effects on body weight development were considered adverse for alcohols, C12-14, ethoxylated, they were not considered adverse for the other three OECD 414 studies in rats due to the limited difference between the treated and control animals. The body weight gain corrected for gravid uterus was slightly reduced compared with control in all four studies, but not considered a toxicologically relevant effect as the difference was limited and not significant.

In the females sacrificed according to the protocol on gestation day 29, prominent lobular architecture of the liver was noted in 1/21, 2/20, 3/21 and 3/21 females in the control, low- mid- and high-dose group, respectively. In addition, 1/21 mid-dose females showed an abnormal appearance of the medial hepatic lobe and 2/21 high-dose females pale discoloration in the liver. The liver changes were considered treatment-related and a result of the metabolic load of the treatment, but not toxicologically relevant.

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

 

Effects on offspring

In the study with alcohols, C12-14, ethoxylated, the mean body weight for male and female fetuses combined was 5, 6 and 8% lower in the low- mid- and high-dose group, respectively, compared with the control.This was considered a treatment-related, but minor, effect and secondary to the systemic maternal toxicity. The changes were therefore not considered adverse. No effects on body weight (gain) were noted in the other study in the rabbit.

A few cases of external malformations were recorded in the two studies in the rabbit, including malrotated hind limbs, cleft palate, hyper-flexed forepaws and malrotated hind paws. Several visceral malformations affecting the subclavian artery, kidneys, heart and major blood vessels, lung, diaphragm, brain and eye as well as skeletal malformations concerning thoracic, lumbar or caudal vertebrae, sternebrae or nasal bones were found. These cases were all considered to be incidental due to the low number or were observed in all groups including control in similar numbers. No treatment-related effects were noted on litter loss, number of corpora lutea, number of pre- and post-implantations, and number of early and late resorptions.

In conclusion, despite the occurrence of ‘episodes’ in the dams, no toxicologically relevant changes were noted in the offspring for any developmental parameters and there was no teratogenicity in the prenatal developmental studies in rabbits.

 

Conclusion on developmental toxicity and teratogenicity in the rat and the rabbit

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 mainly a consistent picture of the effects across the category and species.

Treatment with AE substances according to OECD guideline 422 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 the OECD 422 study with alcohols, C12-15, branched and linear, ethoxylated, the mean body weight of male pups were statistically significantly decreased at PND 7 and 13 in the high-dose group (1000 mg/kg bw/day). In addition, mean body weights of female pups in the high-dose group were decreased at PND 13, though not reaching statistical significance. These findings were considered adverse. However, a significant result was only observed in males at PND 7 and 13 and not in the combined mean body weight for males and females, or for females individually. The reduced weight of male pups is therefore more likely an incidental effect as it is unlikely males put on less weight than females under the same treatment circumstances. No effects on body weight were recorded in the prenatal developmental toxicity study performed with alcohols, C12-15, branched and linear, ethoxylated. As this was the only study among the eight OECD 422 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.

In prenatal developmental toxicity studies performed in rats and rabbits, the maternal effects observed in pregnant rats and rabbits were primarily clinical signs, and reduced body weight development and food consumption, compared with the control. An increased occurrence of ‘episodes’ was recorded in maternal rabbits only, during which the animals showed severe clinical signs that in some cases led to unscheduled sacrifice. No treatment-related and/or toxicologically relevant changes were noted for any other maternal systemic and reproductive parameters recorded in the studies, including: thyroid gland weights (in the rat), thyroid hormone levels (in the rat), microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, pre- and post-implantation sites, pre- and post-implantation loss, viability and litter loss.

In the fetuses, reduced fetal body weight was considered toxicologically relevant in the study with alcohols, C12-14, ethoxylated, however, this was a secondary effect of maternal systemic toxicity. No toxicologically relevant changes were observed in the offspring in any of the remaining developmental parameters, including: litter size, sex ratio, anogenital distance (in the rat) and placental weights of live fetuses. No teratogenic effects were observed in the prenatal developmental studies performed in rats and rabbits.

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 NOAELs for developmental toxicity and teratogenicity are given in Table 2 and 3 above.

For alcohols, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) the following NOAELs are read across:

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 1000 mg/kg bw/day

Oral (rat, OECD 414): NOAEL (teratogenicity) = 1000 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 400 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 400 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, C12-13, branched and linear, ethoxylated (CAS No. 160901-19-9, EC No. 500-457-0) 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.