Alcohols, C12-14, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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

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

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

 

Conclusion based on data obtained with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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:

26 Nov 2019 - 03 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)

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
- Weight at study initiation: males 272-340 g, females 198-249 g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 20-59
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
- Acclimation period: 7 days

IN-LIFE DATES: From 5 FEB 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
- Lot/batch no.: MKCH1635 + MKCG3257, MKCK6411
- Specific gravity: 0.92

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (UPLC-MS ).
- 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. 20218713).

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days.
Females which failed to deliver were treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected based on the results of a 10-day Dose Range Finder (Test Facility Reference No. 20218714) with oral
gavage administration of Alcohols, C12-14, ethoxylated (2 EO) in rats and in an attempt to produce graded responses to the test item.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

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

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

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

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

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in F0 males: testis weight, epididymis weight, 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; possible cause of death was determined for pups born or found dead

Blood was collected from two or three pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

Postmortem examinations (parental animals):

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

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

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, parathyroid, 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) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical
observations and no findings were noted during the weekly arena observations in this study.
In females at 1000 mg/kg bw/day, piloerection was incidentally noted, and uncoordinated
movements and rales were noted once each. Based on the incidence observed this was
considered not to be toxicologically relevant.
Salivation was seen after dosing from 300 mg/kg bw/day upwards in a dose-related trend.
This was considered not to be 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.
Incidental findings that were noted included alopecia, piloerection, abdominal swelling,
abdominal nodule and a broken tail apex. These findings occurred within the range of
background findings to be expected for rats of this age and strain which are housed and
treated under the conditions in this study. At the incidence observed, these were considered
not to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weight and body weight gain were observed up
to 1000 mg/kg bw/day.
A reduced body weight gain was noted for males treated at 1000 mg/kg bw/day during the
first two weeks of treatment. At the end of the study period absolute body weight was 5% lower compared to controls. Based on the magnitude of the change this was considered not to
be toxicologically relevant.
Body weights and body weight gain of males up to 300 mg/kg bw/day and females up to
1000 mg/kg bw/day remained in the same range as controls over the treatment period.

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 recorded.
A slightly lower absolute food consumption was noted during the last week of lactation for
females treated at 1000 mg/kg bw/day (-14% compared to control). Relative food
consumption was reduced from Day 4 of lactation onwards for these animals (up to -12%
compared to control). Based on the magnitude of the change, this was considered not to be
toxicologically relevant.
Any other statistically significant changes in food consumption before or after correction for
body weight were considered to be unrelated to treatment since no trend was apparent
regarding dose and duration of treatment.

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):

Hematology parameters of treated rats were considered not to have been affected by treatment
with the test item.
Any statistically significant changes in hematology parameters were considered to be
unrelated to treatment as these occurred in the absence of a dose-related trend.
Coagulation parameters of treated rats were considered not to have been affected by treatment
with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

An increased alkaline phosphatase (ALP) activity (1.43x of control) and albumin
concentration (1.06x of control) were noted for males treated at 1000 mg//kg/day.
Additionally, a decreased concentration of total bilirubin (up to 0.82x of control) was noted
for males treated at 100, 300 and 1000 mg/kg bw/day, reaching statistical significance for
males at 1000 mg/kg bw/day. For females, an increased concentration of calcium was noted at
300 and 1000 mg/kg bw/day (1.06x of control for both dose levels). Based on the magnitude
of the change, slightly low control values, absence of a dose-related trend and/or as mean
values remained within the historical control ranges2, these changes were considered not to be
toxicologically relevant.
Any other statistically significant changes in clinical chemistry parameters 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 at 1000 mg/kg bw/day were decreased (0.75x of control).
Mean values were within the range of historical control values.
No treatment-related effect was noted on serum levels of T4 in F0-females and serum levels
of TSH in F0-males and females.
Under the conditions of this screening study no associated adverse effect with the decrease in T4 levels in F0 males was observed and so these findings were not taken into account when determining the parental NOAEL.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined
animals up to 1000 mg/kg bw/day. Grip strength was similar between control and treated
animals.
A decreased number of total movements and ambulations (not statistically significant) was
noted for females treated at 1000 mg/kg bw/day. As mean values were within the historical
control range, this was considered not to be toxicologically relevant.
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

Description (incidence and severity):

Test item-related microscopic findings after treatment with Alcohols, C12-14, ethoxylated
(2 EO) were noted at 1000 mg/kg bw/day in the stomach of both sexes, liver and jejunum of
males and adrenal gland of females.
Squamous cell hyperplasia at minimal degree was noted in the forestomach of a single male
and two females at 1000 mg/kg bw/day, shown in Table 2.
Centrilobular hepatocellular hypertrophy of the liver at minimal degree was noted in 4/5
males at 1000 mg/kg bw/day. This was the microscopic correlate to the higher liver weights
and the accentuated lobular pattern recorded at this dose level.Vacuolation of the jejunum was recorded in three males at 1000 mg/kg bw/day. These round,clear vacuoles were present in the lamina propria of the villi of the jejunum, shown in Table 3.
Cortical hypertrophy of the zona fasciculata of the adrenal gland was recorded in 4/5 females
at 1000 mg/kg bw/day at minimal degree, shown in Table 4.
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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item.
Except for one control female which had an irregular cycle, all females had regular
cycles of 4 to 5 days.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The
testis revealed normal progression of the spermatogenic cycle and the expected cell
associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The mating indices were 100, 100, 100 and 90% for the control, 100, 300 and 1000 mg/kg
bw/day groups, respectively.
One animal at 1000 mg/kg bw/day did not show evidence of mating. At this
incidence, the mating index was considered not to be toxicologically relevantly affected.
Precoital time was considered not to be affected by treatment. All mated females showed
evidence of mating within 4 days.
A slightly lower mean number of implantation sites were recorded for animals treated at
1000 mg/kg bw/day (11.8 versus 13.1 in the control group), which was mainly caused by a
slightly lower number of implantation sites for two females. As the change was
minimal and as values remained within the historical control data, this was considered not to
be toxicologically relevant.
Fertility index was considered not to be affected by treatment. The fertility indices were 90,
90, 90 and 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
In every group one mated female was not pregnant. In the absence of a dose-related
incidence, this was considered not to be related to treatment with the test item.
Gestation index and duration of gestation were considered not to be affected by treatment.
All pregnant females had live offspring. The gestation indices were 100% for all groups.
The total number of offspring born compared to the total number of uterine implantations was
considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total
number of uterine implantation sites) was 93, 89, 93 and 93% for the control, 100, 300 and
1000 mg/kg bw/day groups, respectively.

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

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Most pups of litter one litter at 1000 mg/kg bw/day were dehydrated on PND 7, as this occurred on a single day only, this was considered not to be toxicologically relevant.
No clinical signs were noted in other litters at 1000 mg/kg bw/day or at other dose levels.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of
offspring born) was considered not to be affected by treatment. The live birth indices were
100, 100, 98 and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Two pups of a litter at 300 mg/kg bw/day and two pups of a litter 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 remained within the range considered normal
for pups of this age.
Viability index (number of live offspring on PND 4 before culling as percentage of number of
live offspring on PND 1) was considered not to be affected by treatment. Viability indices
were 100, 98, 100, 99% for the control, 100, 300 and 1000 mg/kg bw/day groups,
respectively.
Two pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were missing on PND 3
or 4. Pups missing were most likely cannibalized. No toxicological relevance was attributed
to these missing pups since the mortality incidence did not show a dose-related trend and
remained within the range considered normal for pups of this age.
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, 100, 100, 98% for the control, 100, 300 and 1000 mg/kg
bw/day groups, respectively.
One pup of a litter at 1000 mg/kg bw/day was missing on PND 7. This pup was most
likely cannibalized. No toxicological relevance was attributed to this missing pup since the
mortality incidence remained within the range considered normal for pups of this age

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slightly lower body weight gain was noted for pups at 1000 mg/kg bw/day, (-10% for
males and females combined on PND 13; not statistically significant). This was mainly
caused by the lower body weight of one litter. Based on the magnitude of the change and
as the body weights of other litters were in the range of control litters, this was considered not
to be toxicologically relevant.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Serum T4 levels of PND 14-16 pups were increased (1.23x and 1.22x of control for males and
females, respectively) at 1000 mg/kg. Serum T4 levels of PND 14-16 pups at 100 and 300
mg/kg bw/day remained in the same range as controls. However, under the conditions of this screening study no associated adverse effect was observed and so these findings were not taken into account when determining the developmental NOAEL.
No test item-related effect was noted for serum TSH levels of PND14-16 pups or serum T4
levels in PND4 pups.

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.
The statistically significantly higher mean corrected anogenital distance for female pups at
1000 mg/kg bw/day was considered not to be toxicologically relevant in absence of a dose
related trend.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically relevant effects obseerved

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1
Mean Percent Liver and Kidney Weight Differences from Control Groups

 

Dose level (mg/kg/day):	100			300		1000
LIVER (Males)
Absolute	11			24**		37**
Relative to body weight	7			18**		40**
KIDNEY (Males)
Absolute	9			13*		11*
Relative to body weight	5			8		13**
ADRENAL GLANDS (Females)
Absolute	7		-3				27*
Relative to body weight	4		0				27*

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

Table 2
Summary Test Item-Related Microscopic Findings – Both sexes

	Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
STOMACH ^a	5	5	5	5	5	5	5	5
Hyperplasia, squamous cell,                                  forestomach
Minimal	-	-	-	1	-	-	-	2

^a  =  Number of tissues examined from each group.

Table 3
Summary Test Item-Related Microscopic Findings – Males

	Males
Dose level (mg/kg/day):	0	100	300	1000
LIVER ^a	5	6	6	5
Hypertrophy, hepatocellular,                                 centrilobular
Minimal	-	-	-	4
JEJUNUM ^a	5	5	5	5
Vacuolation
Minimal	-	-	-	3

^a  =  Number of tissues examined from each group.

Table 4
Summary Test Item-Related Microscopic Findings – Females

	Females
Dose level (mg/kg/day):	0	100	300	1000
ADRENAL GLAND ^a	5	5	5	5
Hypertrophy cortical,                             zona fasciculata
Minimal	-	-	-	4

^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:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Alcohols, C12-14, ethoxylated

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies:
With respect to reproductive toxicity, only a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test according to OECD guideline 422 is available with the registered substance. In this study, no relevant effects in relation to reproductive and developmental toxicity have been observed. In addition, a subchronic (90-day) oral repeated dose toxicity study, a prenatal developmental toxicity study in rats and a prenatal developmental toxicity study in rabbits, all conducted with the registered substance, did not reveal any observations in reproductive tissues and organs indicative of reproductive / developmental toxicity.
- Available non-GLP studies:
No study to meet the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance is available.
- Historical human data:
The registrant is not aware of any relevant human data suitable to satisfy the information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 with the registered substance.
- (Q)SAR:
No QSARs are available to reliably predict the parameters assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, a QSAR assessment is not possible.
- In vitro methods:
No in vitro methods are available to reliably predict the parameters assessed in an extended one-generation reproductive toxicity study (OECD 443). Therefore, in vitro studies are not possible.
- Weight of evidence:
No adequate and reliable data are available that possibly could be used for a Weight-of-Evidence assessment of reproductive toxicity for the registered substance.
- Grouping and read-across:
The registered substance is a member of the Alcohol Ethoxylates (AE) category. The category contains structurally similar substances sharing a similar toxicokinetic behaviour and similar toxicological properties. In fact, robust read-across approaches are applied for several human health-related endpoints, including systemic and local toxicity. However, only four of the AE substances are subject to information requirements according to Annex X of the REACH Regulation (EC) No. 1907/2006 and no adequate and reliable studies concerning reproductive toxicity (other than screening studies) are available in the database of the category. It is intended to perform extended one-generation reproductive toxicity studies (OECD 443) for two of the four substances and to apply a read-across approach to cover the information requirement of Annex X, Section 8.7.3. for the remaining two substances. Based on the structural properties of the registered substance, it has been selected as one of the source substances. In consequence, the only way to comply with the standard information requirement of Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 is to perform an extended one-generation reproductive toxicity study according to OECD guideline 443 with the registered substance.
- Substance-tailored exposure driven testing:
The registered substance is used in a variety of applications. None of the conditions and prerequisites of Annex XI, Section 3. of the REACH Regulation (EC) No. 1907/2006 can be met. Therefore, substance-tailored exposure driven testing cannot be applied for the registered substance.
- Approaches in addition to above:
Not applicable
- Other reasons:
Not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Annex X, Section 8.7. (Reproductive Toxicity), Column 2, of the REACH Regulation (EC) No. 1907/2006 stipulates the following specific adaption options:
‘The studies need not be conducted if:
• the substance is known to be a genotoxic carcinogen, meeting the criteria for classification both in the hazard class germ cell mutagenicity (category 1A or 1B or 2) and carcinogenicity (category 1A or 1B), and appropriate risk management measures are implemented, or
• the substance is known to be a germ cell mutagen, meeting the criteria for classification in the hazard class germ cell mutagenicity (category 1A or 1B) and appropriate risk management measures are implemented, or
• the substance is of low toxicological activity (a comprehensive and informative dataset showing no toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure.
If a substance is known to have an adverse effect on sexual function and fertility, meeting the criteria for classification in the hazard class reproductive toxicity (category 1A or 1B: May damage fertility (H360F)), and the available data are adequate to support a robust risk assessment, then no further testing for sexual function and fertility shall be necessary.
If a substance is known to cause developmental toxicity, meeting the criteria for classification in the hazard class reproductive toxicity (category 1A or 1B: May damage the unborn child (H360D)), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity shall be necessary.’
None of the above-mentioned conditions is met. Therefore, the specific Column 2 adaption options are not adequate to generate the necessary information for the registered substance.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
The testing proposal is based on the results of a screening study for reproductive / developmental toxicity according to OECD guideline 422 and the lack of relevant effects in reproductive tissues and organs indicative of reproductive toxicity observed in subchronic (90-day) oral repeated dose toxicity (OECD 408) and prenatal developmental toxicity studies in rats and rabbits (OECD 414).
The following study is proposed: Extended one-generation reproductive toxicity study according to Annex X, Section 8.7.3., of the REACH Regulation (EC) No. 1907/2006 (B.56 of the Commission Regulation on test methods or OECD guideline 443) in rats, oral route with the registered substance, specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation
- Dose level setting shall aim to induce some toxicity at the highest dose level
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation
For a justification of the test design, please refer to ‘Justification of study design’ below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals:
Ten weeks premating exposure duration for the parental (P0) generation as recommended by ECHA. This is the default value as specified by ECHA in recent decisions on OECD 443 studies.
- Basis for dose level selection:
The dose level setting shall aim to induce some toxicity at the highest dose level but not causing suffering or death. A recently performed combined repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422), a subchronic (90-day) oral repeated dose toxicity (OECD 408) and a prenatal developmental toxicity (OECD 414) studies in rats with the registered substance are available. Dose levels applied in the studies were 100, 300 and 1000 mg/kg bw/day. Whilst no systemic toxicity was observed in the combined repeated dose toxicity and the subchronic (90-day) oral repeated dose toxicity studies, treatment with the test substance resulted in reduced maternal body weights considered adverse in the prenatal developmental toxicity study. The No-Observed-Adverse-Effect-Level (NOAEL) in the latter study was set at the mid-dose level of 300 mg/kg bw/day.
- Inclusion/exclusion of extension of Cohort 1B:
This cohort is a standard requirement of an OECD guideline 443 study. Cohort 1B (Reproductive toxicity) will be included without extension to mate the Cohort 1B animals to produce the F2 generation.
Based on the available combined repeated dose toxicity study with the reproduction / developmental toxicity screening test (OECD 422), subchronic (90-day) oral repeated dose toxicity study (OECD 408) and prenatal developmental toxicity studies (OECD 414) in rats and rabbits, all conducted with the registered substance, no relevant effects or adverse changes in reproductive tissues and organs indicative for reproductive / developmental toxicity have been observed. Therefore, the extension to mate the Cohort 1B animals is not justified.
- Termination time for F2:
Not relevant as the production of the F2 generation is not planned.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B:
The Cohorts 2A and 2B are not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 2A and 2B.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3:
The Cohort 3 is not planned to be included in the study design. There are no adverse effects in relation to reproductive / developmental toxicity observed in any of the studies mentioned above which might trigger the inclusion of Cohort 3.
- Route of administration:
The standard route of administration is the oral route.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals:
The standard species is the rat. It is intended to use the same rat strain as used in previous studies (Crl: WI(Han)) depending on the historical control database in the selected test facility. All other parameters will be selected as recommended in OECD guideline 443.

Species:

rat

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies performed with member substances of the Alcohol Ethoxylates (AE) category including data on the target substance. The selected studies are 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, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) as well as several member substances of the Alcohol Ethoxylates (AE) category.

 

Study with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3)

The reproductive toxicity of alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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, 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 days whereas females that delivered were treated for 50-54 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 42-51 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.

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs). Except for one control female which had an irregular cycle, all females had regular cycles of 4 to 5 days. The mating indices were 100, 100, 100 and 90% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One animal treated at 1000 mg/kg bw/day did not show evidence of mating. At this incidence, the mating index was considered not to be adversely affected. Precoital time was considered not affected by treatment as all mated females showed evidence of mating within 4 days. A slightly lower mean number of implantation sites were recorded for animals treated at 1000 mg/kg bw/day (11.8 versus 13.1 in the control group), which was mainly caused by a slightly lower number of implantation sites for  two females. As the change was minimal and as values remained within the historical control data, this was considered not to be toxicologically relevant. Fertility index was considered not to be affected by treatment. The fertility indices were 90, 90, 90 and 89% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. In every group one mated female was not pregnant. In the absence of a dose-related incidence, this was considered not to be related to treatment with the test item. Gestation index and duration of gestation were considered not to be affected by treatment; all pregnant females had live offspring. The gestation indices were 100% for all groups. No signs of difficult or prolonged parturition were noted among the pregnant females.

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

Studies in the AE category

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

Table 1

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

Conclusion ontoxicity to reproduction (fertility)

The data available for alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) is consistent with the overall toxicity to reproduction data for AE substances. The following NOAELs were set: 

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

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

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

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

Effects on developmental toxicity

Description of key information

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 300 mg/kg bw/day

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

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 200 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 200 mg/kg bw/day

 

Conclusion based on data obtained with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Aug - 10 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

no

Principles of method if other than guideline:

The females of each group were divided in one of 4 subgroups, depending on the Day of coitum, and dosed accordingly. Based on 14/21 early deliveries in Subgroup 2, along with high fetal weights for this subgroup, and low fetal weights for Subgroup 1, it is suspected that Subgroups 1 and 2 may have been switched after mating.
This would indicate that animals of Subgroup 1 (Female Nos. 1-6 (control), 23-27 (100 mg/kg bw/day), 45-49 (300 mg/kg bw/day), 67-72 (1000 mg/kg bw/day)) were dosed on Days 5-19 post-coitum and sacrificed on Day 20 post-coitum, and animals of Subgroup 2 (Female Nos. 7-11 (control), 28-33 (100 mg/kg bw/day), 50-55 (300 mg/kg bw/day), 73-77 (1000 mg/kg bw/day)) were dosed on Days 7-21 post-coitum and sacrificed on Day 22 post-coitum. Subgroups 3 and 4 were dosed according to schedule (i.e., dosing on Days 6-20 post-coitum with euthanasia on Day 21 post-coitum). 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.
Humidity was outside the target range on 4 days (maximum of 80%) without a noticeable effect on the clinical condition of the animals.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wl(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 - 15 weeks
- Weight at study initiation: 179 - 267 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 (%): 57 - 80
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Aug 2021 To: 10 Sep 2021

Route of administration:

oral: feed

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 me asurement and the dose formulations were stirred continuously during dosing.

VEHICLE
- Concentration in vehicle: 25 mg/mL (for 100 mg/kg bw/day group), 75 mg/mL (for 300 mg/kg bw/day group), 250 mg/mL (for 1000 mg/kg bw/day group),
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted in Week 1 and 2 to assess accuracy and homogeneity.

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
A small response at the retention time of the ammonium adduct of the C12E6 compound was observed in one of the chromatograms of the Group 1 formulation prepared for use in Week 1. It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. In the other formulation of Group 1, no test item was detected.

The formulations of Group 2 and Group 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 gestation is the day of mating and is referred to as Day 0 post-coitum.

Duration of treatment / exposure:

Days 6 - 20 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy on Day 21 post-coitum

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

1 000 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, 1000 mg/kg bw/day, in consultation with the Sponsor and based on results of a Combined 28-day repeated dose toxicity study with Reproduction/Developmental Toxicity Screening Test with oral exposure of the test substance in rats (Test Facility Study No. 20218715).
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy performed directly post dosing. Mortality and Morbidity were checked twice daily.

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

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

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 coitum.

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-coitum
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 comparis ons 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 regarding embryo-fetal development was provided and can be found in Attachment 3 under "Overall Remarks, Attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 100 mg/kg bw/day group, the only clinical sign observed was hunched posture in 1/22 female in the days pre-dosing (Day 2 post coitum). It was therefore not considered treatment-related.

Hunched posture was observed for 2/22 females of the 1000 mg/kg bw/day group on some occasions between Days 7-21 post-coitum and for 1/22 females of the 300 mg/kg bw/day group between Days 9-18 post-coitum. In the high dose group, erected fur was noted for 4/22 females on several occasions and 1/22 female had abnormal breathing sounds on Day 16 post-coitum. These signs were considered treatment-related but not adverse.

Salivation was seen on several occasions in 9/22 and 5/22 females of the 1000 and 300 mg/kg bw/day group, respectively. This 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).

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

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The differences in body weight have been evaluated based on the different subgroups.
At 100 mg/kg bw/day, mean body weights, body weight gain and weight gain corrected for gravid uterus were comparable to control. At 300 mg/kg bw/day, body weight gain and body weight gain corrected for gravid uterus weight was slightly lower in Subgroup 1 only (21 vs 38 g in concurrent controls, the difference in adjusted body weight gain reached statistical significance).
In the 1000 mg/kg bw/day group, all subgroups showed toxicologically relevant differences to the control. Body weight gain for Subgroups 3+4 was lower throughout the dosing period, with a total mean body weight gain of 37% vs 48% in concurrent control. A similar effect on body weight gain was observed in Subgroup 1 (35% vs 47% in the control) and in Subgroup 2 (22% vs 37% in concurrent control). It has to be noted that for Subgroup 2, the body weight of Day 18 post-coitum was used as the last time point for evaluation as several animals of this subgroup delivered early.
Terminal body weight was 11%, 7% and 7% lower compared to the control for Subgroup 1, 3 and 4, respectively. In Subgroup 2, mean body weight of animals that delivered early was 4% lower than control at the end of the dosing period. For the two females in Subgroup 2 that did not deliver early, mean body weight was 9% lower than concurrent control on Day 21 post-coitum.
Slight weight loss was observed in several animals of the 1000 mg/kg bw/day group during the study: 1 female of Subgroup 2 (Days 6 - 9 post-coitum), 3 females of subgroup 1 (Days 6 - 9 post-coitum) and 4 females in Subgroup 3+4 (Days 6 - 9 post-coitum).
At 1000 mg/kg bw/day, body weight gain corrected for gravid uterus weight was lower: 15 vs 38 g and 6 vs 26 g in Subgroups 1 and 3+4, respectively compared to concurrent controls. For Subgroup 2, body weight gain corrected for gravid uterus weight could not be evaluated due to the early delivery of several females of this subgroup.

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

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake at 100 mg/kg bw/day was considered comparable to control in all subgroups.
At 300 mg/kg bw/day, food intake for Subgroup 1 animals was reduced during the complete dosing period. The mean overall food intake was -11% compared to the control, but the difference did not reach statistical significance. Food intake in the remaining subgroups at 300 mg/kg bw/day was considered comparable to control. Because of the sporadic finding in only one subgroup and the minor difference, this finding was not considered adverse at 300 mg/kg bw/day.
In the high dose group, food consumption was reduced in all subgroups during the complete dosing period. For most of the intervals in Subgroup 1 and 3+4, these differences reached statistical significance. In Subgroup 2, food consumption was also decreased in all intervals with the exception of Day 18 - 21 post coitum, but none of the differences reached statistical significance. For Subgroups 1, 2 and 3+4, mean overall food intake was 24, 11 and 17% lower than concurrent controls, respectively. For Subgroup 1 and 3+4, this difference was statistically significant.

Summarized results can be found in Attachment 2 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 was observed regarding water consumption between the control and treatment groups up to and including the highest dose level.

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 applicble

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant difference was observed regarding thyroid hormones between the control and 100 mg/kg bw/day group.

At 300 and 1000 mg/kg bw/day, total T3 concentration was decreased (0.86x (0.388 ng/mL) and 0.79x (0.356 ng/mL) of control (0.451 ng/mL), respectively) and TSH levels were increased (1.31x (0.3421 mU/L) and 1.66x (0.4347 mU/L) of control (0.2619 mU/L), respectively). The difference in TSH level in the 300 mg/kg bw/day group did not reach statistical significance. However, all mean values were within the available historical control data range, therefore, they were considered non-adverse.

The historical control data ranges were (2020 - 2021):
T3 (ng/mL) mean: 0.439; P5 - P95: 0.280 – 0.616 (n = 263)
TSH (mU/L) mean: 0.353; P5 - P95: 0.127 – 0.699 (n = 373)

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

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

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):

No toxicologically relevant difference was observed regarding thyroid weights between the control and treatment groups up to and including the highest dose group.

In the 1000 mg/kg bw/day group, thyroid weights were decreased compared to the control group (-16%), but this change was considered within biological variation as it was small and no associated histopathological findings were noted.

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant difference was observed regarding gross pathology between the control and treatment groups up to and including the highest dose group.

A pale discoloration of the liver was noted in 3 females (2/22 of the 300 mg/kg bw/day group, 1/22 of the 1000 mg/kg bw/day group). The liver of the female of the high dose group was also enlarged. 1/22 females of the high-dose group had a a thick, non-glandular stomach and 1/22 females in the 300 mg/kg bw/day group had enlarged (right) and smaller (left) thyroid gland. Given the incidental nature of these findings, no association with histopathology and/or the absence of a dose-related response, these findings were considered unrelated to treatment with the test item.

Summarized results 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):

There were no test item-related microscopic observations in the thyroid glands.

Summarized results 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 abortion occurred in this study.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No toxicologically relevant difference was observed regarding pre- and post-implantation loss between the control and treatment groups up to and including the highest dose group.

Pre-implantation loss was slightly higher for females at 1000 mg/kg bw/day group compared to the control group (10.38% vs 7.44% in control). However, as the difference was not statistically significant, mean values remained within the available historical control data (mean: 6.5; P5 - P95: 2.2 – 11.8 (n = 1295) in a period from 2016 to 2020) and individual values were within the concurrent control range, this was considered not related to treatment with the test item.

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

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no total litter losses by resorption in the control or treatment groups up to and including the highest dose group.

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

Early or late resorptions:

no effects observed

Description (incidence and severity):

No difference was observed regarding resorptions between the control and treatment groups up to and including the highest dose group.

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

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses were observed in any of the groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No toxicologically relevant difference was observed between the control and treatment groups up to and including the highest dose level.

3 control females (Nos. 7, 8, 11), 5 females at 100 mg/kg bw/day (Nos. 29, 30, 31, 32, 33), 4 females at 300 mg/kg bw/day (Nos. 50, 51, 53, 54) and 2 females at 1000 mg/kg bw/day (Nos. 74, 76), all in Subgroup 2, delivered their litter on the day of scheduled necropsy. These females were examined according to scheduled necropsy and included in the evaluation. These early deliveries are not considered treatment-related but caused by the unintentional switch of subgroups, as they occurred in Subgroup 2 only and across all groups, including the control.

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

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No statistically significant difference was observed regarding the number of pregnant animals between the control and treatment groups up to and including the highest dose group. The numbers of females with viable litters for evaluation were 21, 21, 21 and 22 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Summarized results 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:

systemic toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects were observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day mean fetal body weight was within the same range as concurrent controls for all subgroups.
At 300 mg/kg bw/day, mean fetal body weight (male, female and combined) in Subgroup 1 was slightly lower (4% lower than control for combined weights), but the difference did not reached statistical significance and was therefore not considered adverse.
At 1000 mg/kg bw/day, the mean fetal body weights was compared according to subgroup. In Subgroup 1 and 3+4 (male, female and combined) mean fetal body weights were lower compared to the control group (combined weights -8% and -4% in Subgroup 1 and 3+4, respectively), but the difference did not reach statistical significance. Mean fetal body weights at the high dose in Subgroup 4 were below or at the lower end of the historical control range. For Subgroup 2, mean fetal body weights (male, female and combined) was in the same range as the concurrent control.

It has to be noted that fetal body weight for all dose groups of Subgroup 1 (including control) was below the lower range of the available historical control data, while fetal body weight for all
dose groups of Subgroup 2 (including control) was above the upper range of the available historical control data. This was attributed to the suspected switch of Subgroup 1 and 2; fetuses were likely weighed on Day 20 post-coitum in Subgroup 1 and on Day 22 post-coitum in Subgroup 2. Fetal body weight of control Subgroup 3+4 was within the historical control data (5.21 g for males and 5.08 g for females).

Historical control data for body weight of Wistar Han fetuses (period 2016-2020):
Mean male body weight (g) mean: 5.4; P5 - P95: 5.2 – 5.5 (n = 6774).
Mean female body weight (g) mean: 5.1; P5 - P95: 4.9 – 5.3 (n =7024).
Mean fetal body weight (g) mean: 5.3; P5 - P95: 5.0 – 5.4 (n = 13798).

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

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no differences regarding the number of live fetuses between the control and treatment groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment with the test item up to
1000 mg/kg bw/day. Mean sex ratios (males:females) were 49:51, 50:50, 57:43 and 53:47 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

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

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size of any group.
Mean litter sizes were 11.7, 12.2, 11.8 and 11.2 fetuses/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

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

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean anogenital distance (AGD) in male and female fetuses (absolute and normalized for fetal body weight) was considered comparable to control for all treated groups.

The mean anogenital distance for male fetuses of Subgroup 1 of all dose groups (including control) was below the lower range of the available historical control data (1.447 - 1.514), but within the historical control range for the female fetuses. The mean anogenital distance was within the available historical control data for Subgroups 2 and 3+4 for both sexes.


Historical control data for Anogenital distance of Wistar Han fetuses (period 2016-2020):
Male anogenital distance (mm) mean: 2.8; P5 - P95: 2.6 – 3.3 (n = 6774).
Female anogenital distance (mm) mean: 1.3; P5 - P95: 1.1 – 1.7 (n = 7024).

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

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

no effects observed

Description (incidence and severity):

There were no external malformations and variations observed in any groups.

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

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Three skeletal malformations were noted: the thoracic vertebral column and ribs were affected in one fetus of the 300 mg/kg bw/day and in one fetus of the 1000 mg/kg bw/day group, one fetus of the control group had a supernumerary lumbar vertebra. These cases were considered incidental.

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; statistically significant)) were noted at higher incidences at 1000 mg/kg bw/day, although most were not statistically significant. These changes are variations, not malformations and are not considered adverse, but as a pattern was recognized, it will be described here for completeness. To describe the results, the observations for the 4 subgroups have to be viewed separately.

In Subgroup 1, delays of ossification were particularly shown at 1000 mg/kg bw/day (unossified metacarpals and sternebrae and incompletely ossified vertebral centra and arches) and at 300 mg/kg bw/day (unossified sternebrae) and 100 mg/kg bw/day (unossified metacarpals).

In Subgroup 2, fetal skeletal ossification was more advanced than for the fetuses of subgroup 1, which was associated with the higher fetal weights in this subgroup. No ossification delays were noted.

In Subgroup 3+4, a low incidence of incompletely ossified skull bones (e.g., interparietal and parietals) was observed without any test item-relationship between the groups. This implies a more advanced ossification of the fetuses than observed in the fetuses of Subgroup 1, but a poorer ossification when compared to fetuses of Subgroup 2.

In groups treated with the test item, statistically significantly fewer fetuses had wavy ribs when compared to the control group. This is not considered to be treatment-related.

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

Visceral malformations:

no effects observed

Description (incidence and severity):

No toxicologically relevant malformations or variations were observed in any of the control and treatment groups. Only one malformation, which was considered incidental, occurred in one fetus of the 300 mg/kg bw/day group. This fetus had a small eye.

The variations that were noted affected the liver (supernumerary lobes), ureters (convoluted) and urinary bladder (distended) at low incidences or in isolated cases, which did not indicate any test item-relationship.

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

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

300 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 this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

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 Wistar Han rats at levels up to 300 mg/kg bw/day. The maternal systemic and developmental No Observed Adverse Effect Levels (NOAELs) for the test substance were set at 300 mg/kg bw/day, based on the effects on maternal and fetal body weights. No teratogenicity was observed at any dose level.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 16 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose range finder 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:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 19 - 20 weeks old
- Weight at study initiation: the group means on Day 7 (start of dosing) were 2944 - 4383 g
- Fasting period before study: not applicable
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet: KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Granovit AG, Kaiseraugst, Switzerland (25 g at arrival, on subsequent days 140 - 160 g). In addition, pressed hay (daily allotment; Tecnilab-BMI B.V., Someren, The Netherlands) and fresh fruits and/or vegetables (at least twice weekly) were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: 5 - 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

In Life Dates: From: 24 Jan 2022 To: 18 Feb 2022

Route of administration:

oral: gavage

Vehicle:

propylene glycol

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.

VEHICLE
- Concentration in vehicle: 20 mg/mL (30 mg/kg bw/day), 53.33 mg/mL (80 mg/kg bw/day), 133.33 mg/mL (200 mg/kg bw/day)
- Amount of vehicle (if gavage): 1.5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20298606) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
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 90-110% of target concentration).
The formulations of Group 2 and Group 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. The day of mating was assumed to be the first day of gestation and was referred to as Day 0 post-coitum.

Duration of treatment / exposure:

Day 7 to Day 28 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy at Day 29 post-coitum

Dose / conc.:

30 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

80 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

200 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 were selected in consultation with the Sponsor and based on the results of a dose range finder study in pregnant rabbits with oral exposure to the test substance, Test Facility Study No. 20298604), and in an attempt to produce graded responses to the test item. In this study, pregnant rabbits were exposed to the test substance via oral gavage at dose levels of 200, 300, and 400 mg/kg bw/day. In total, thirteen animals (six at 400 mg/kg bw/day, six at 300 mg/kg bw/day and one at 200 mg/kg bw/day) did not survive until scheduled necropsy. All these deaths were considered test-item related; four animals (one, one and two at respectively 200, 300 and 400 mg/kg bw/day) were sacrificed in extremis as the humane endpoint was reached, based on severe clinical signs of toxicity, highly suspected to be of neurological origin. These signs included: increased respiratory rate, shallow breathing, prostrate, hunched posture, both eyes completely or partially closed, severely uncoordinated, lying on side, extensor rigidity of both hindlimbs, decreased activity, head tilt and/or lack of/impaired righting reflex. In addition, nine animals (five and four at respectively 300 and 400 mg/kg bw/day) were sacrificed on veterinary advice (also see Attachment B for a veterinary statement regarding study 20298604) as these animals presented with similar clinical signs of toxicity and since the remainder of the group was sacrificed in extremis, sufficient data was obtained to considered 300 and 400 mg/kg bw/day too high for dosing in the Main study. Moreover, lower food consumption was observed from 200 mg/kg bw/day onwards, with apparent recovery by the end of the treatment period at 200 mg/kg bw/day only. At 300 mg/kg bw/day, slight body weight loss was noted in 3/6 animals between Day 9-12 post-coitum and for 2/6 animals between Day 12-15 post-coitum, followed by stable body weight gain throughout the remainder of the treatment period. Body weight gain at 200 and 400 mg/kg bw/day was considered stable. With the exception two animals (on each at 200 and 300 mg/kg bw/day), all animals were pregnant at (early) termination. As all animals at 300 and 400 mg/kg bw/day were sacrificed prior to Day 29 post-coitum, there was no data available to investigate remaining maternal and fetal parameters at these dose levels. At 200 mg/kg bw/day, the number of corpora lutea was lower, as well as the number of implantations. Pre- and post-implantation loss was increased, which was caused by a higher number of early resorptions.

Based on the abovementioned findings and in consultation with the Sponsor, the following dose levels were selected: 30, 80 and 200 mg/kg bw/day, as clearly higher doses could not be tolerated by the animals.

Moreover, based on the timing of the observed clinical signs, a second post-dose clinical observation at 2 hours after dosing was added to the in-life procedures and criteria were set up by the veterinarian team to define the humane endpoint based on the observed clinical signs in the dose range finder study (Test Facility Study No. 20298604).

Maternal examinations:

CHECK FOR MORTALITY: Yes
- Time schedule: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily; starting on Day 7 post-coitum up to the day prior to necropsy. Observations were performed 1 hour (+/- 15 minutes) and 2 hours (+/- 15 minutes) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum, body weight gains were calculated for the intervals Days 7 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, 24 - 27, 27 - 29, and 7 - 29 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum
- Organs examined: Animals were sacrificed by intravenous injection of sodium pentobarbital. All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in the appropriate fixative.

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: Yesa
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes

Blood sampling:

No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter (heads of ~50% of fetuses were fixed in Bouin’s solution for soft-tissue examination, tissues without variations or malformations were discarded. Tissues with variations or malformations were stored. Heads of other ~50% of fetuses were examined by midcoronal
slice)
- Body weight: Yes (all per litter of viable and non-viable fetuses surviving until scheduled necropsy on Day 29 post-coitum)
- Sex determination: Yes (all per litter of viable and non-viable fetuses surviving until scheduled necropsy on Day 29 post-coitum)

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were 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 regarding maternal pregnancy data and fetal pathology was provided and can be found in Attachment 3 under "Overall remarks, attachments".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the low dose group, no treatment-related clinical signs were observed.
At 80 mg/kg bw/day, decreased activity was noted in 2/22 animals on Day 12 post-coitum, 1 h post-dose, as well as in 7/22 animals on individual or multiple days between Days 21 - 28 post-coitum. Besides that, 1/22 females was observed with thin appearance between Days 26 and 28 post-coitum. Although test material-related, based on the milder nature of these findings compared to the high dose group and as they were considered transient, the clinical signs at 80 mg/kg bw/day were considered not adverse.
In the 200 mg/kg bw/day group, behavioral episodes were noted on several (consecutive) days of treatment from Day 15 post-coitum onwards in 5/22 animals (including the animal that was sacrificed early). These episodes consisted of the following clinical signs: increased respiratory rate, shallow breathing, severely uncoordinated movements, lying on side, headtilt, prostrate, decreased activity and/or extensor rigidity of both hindlimbs, prior to and/or during the first post-dose observation. Moreover, decreased activity was noted in up to all animals from Day 8 post-coitum onwards, during 1 and/or 2 hour post-dose observations. In addition, uncoordinated movement (up to moderate) and/or lying on side was noted in respectively 19 and 16 of these animals, also during 1 and/or 2 hour post-dose observations from respectively Days 8 and 10 post-coitum. Additionally, in individual animals, hunched posture, erected fur, prostrate (in the absence of an episode) and/or partially closed eyes were noted on multiple occasions between Days 15 and 28 post-coitum.

Summarized results 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):

The following premature deaths were noted:
- Control group: 1/22 animals
- 30 mg/kg bw/day: 2/22 animals
- 80 mg/kg bw/day: 1/22 animals
- 200 mg/kg bw/day: 1/22 animals

No dose-relationship was observed.

Details on premature deaths:
The animals of the control group was sacrificed in extremis for animal welfare reasons on Day 17 post-coitum as it had limited to absent food consumption for 7 consecutive days, which is considered the humane endpoint. Slight body weight loss was noted for this animal on Day 15 (3% compared to the highest recorded body weight). No clinical signs were noted for this animal. At necropsy, ectopic splenic tissue and clear watery cysts on the oviducts were noted. These findings are occasionally seen among rabbits used in these types of study and were considered changes of no toxicological significance. This animal was pregnant with normal implantation sites for the duration of pregnancy.

1 of the 2 animals of the 30 mg/kg bw/day group sacrificed prematurely was sacrificed in extremis for animal welfare reasons on Day 8 post-coitum as it did not acclimatize to the facility properly. Since its arrival at the Test Facility, the animal showed (almost) absent food consumption. Also, between Days 3 (first body weight recorded on site) and 7 (start dosing) the animal lost 4% of its body weight. The animal was noted with some type of abnormality on its left eyelid, causing a swollen eyelid, red discoloration of the eyeball and clear as well as mucoid discharge from the eye. At necropsy, it was noted with a white nodule with hair on the sclera of the left eye, as well as prominent lobular architecture of the liver. This animal was pregnant with normal implantation sites for the duration of pregnancy. As the poor condition of this animal was observed already during the acclimation period was considered to be unrelated to treatment with the test material.
The other animal of this dose group was sacrificed in extremis for animal welfare reasons on Day 21 post-coitum as it was noted with low to absent food consumption for 8 consecutive days. In addition, moderate body weight loss was noted for this animal (6% compared to the highest recorded body weight on site). No clinical signs were noted for this animal. At necropsy, prominent lobular architecture of the liver was noted. This animal was pregnant with normal implantation sites for the duration of pregnancy. As these effects were similar to those observed in an animal of the control group, the death was regarded as incidental.

The animal of the 80 mg/kg bw/day group was sacrificed as per protocol as it started to deliver its litter early on Day 24 post-coitum. On day 23 post-coitum, erected fur and decreased activity were noted for this animal. Except for slight body weight loss between Days 12-15 post-coitum, normal body weight gain was noted for this animal. Stable food consumption was noted for this animal, with the exception of the two days prior to its delivery, during which low to absent food consumption was noted. At necropsy, no macroscopic abnormalities were noted. This female had 3 dead fetuses, 1 early resorption and 7 late resorptions. Although delivery of a litter on Day 24 post-coitum occurs very rarely in rabbits, based on the single incidence of this early delivery at the mid dose level only, it was considered not related to treatment with the test material.


The animal of the 200 mg/kg bw/day group was sacrificed in extremis for animal welfare reasons on Day 17 post-coitum as it presented with a behavioral episode on three consecutive days of treatment (Days 15, 16 and 17 post-coitum). These behavioral episodes consisted of the following clinical signs: increased respiratory rate, severely uncoordinated movements, lying on side, headtilt, prostrate, hunched posture, decreased activity and/or extensor rigidity of both hindlimbs, prior to and/or during the first and/or second post-dose observation. Body weight (gain) for this animal was considered within normal range. Normal food consumption was noted for this animal up to Day 12 post-coitum and lower food consumption from Day 12 onwards. At necropsy, ectopic splenic tissue was noted, which is a finding that is occasionally seen among rabbits used in these types of study and were considered changes of no toxicological significance. This animal was pregnant with normal implantation site for the duration of pregnancy. The early sacrifice of the 200 mg/kg bw/day animal was considered test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the low- and mid-dose group the overall mean body weight gain (gestation day 7 - 29) was decreased by 9 and 11%, respectively, compared to the control group (not statistically significant). In the high-dose group the overall mean body weight gain (gestation day 7 - 29) was decreased by 12% compared to the control group (not statistically significant). As the overall change was minimal for all groups and the intervals (e.g. gestation day 7 – 9) showed both a higher and a lower body weight gain than the control for all treatment groups, this was not considered an adverse effect.
Body weight gain corrected for gravid uterus weight was lower in all treated groups, compared to control. A higher mean body weight loss was recorded for all treated groups compared to the control group, when adjusting the total body weight changes for the gravid uterus weight (-117.4, -141.8 and -151.2 grams at 30, 80 and 200 mg/kg bw/day, respectively, vs. -91.8 grams in the control group). Based on the small magnitude of the effect, this was considered not adverse.

Summarized results 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 all treated groups, although not statistically significant, mean food consumption was lower compared to controls throughout most of the treatment period.
Overall mean food consumption between Days 7-29 post-coitum was dose-dependently lower in all treated groups (-3.27, -6.49, and -7.48% lower compared to controls at 30, 80, 200 mg/kg bw/day, respectively). Based on the small magnitude of the change (<10%), this finding was considered not adverse.

Summarized results 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 was observed between the control and treatment groups.

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:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg bw/day, gross findings observed at necropsy were considered to be unrelated to treatment with the test material.
In the 80 mg/kg bw/day group, liver findings were noted in 2/21 females (prominent lobular architecture in 2/21 females and abnormal appearance of the medial lobe in 1/21 females). In addition, decreased adipose tissue was noted (correlating to thin appearance) in a single female at 80 mg/kg bw/day, together with a misshapen spleen. Based on the single incidence of these findings, it was considered a chance finding rather than a test material-related effect.

At 200 mg/kg bw/day liver findings were noted in 3/21 females (prominent lobular architecture in 3/21 females, and pale discoloration in 2/21 females). 1/21 females had a small thymus.

As the liver findings were also observed in the animals that were sacrificed early, they are considered treatment-related but non-adverse.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

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 were observed during the study.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The pre- and post-implantation losses (%) were unaffected by treatment with the test item up to 200 mg/kg bw/day.

The increased pre-implantation loss observed in the concurrent control group was caused by an individual female which had a pre-implantation loss of 90.9% [historical control data – mean (P5-P95: 6.1 (1.6-12.7) – based on a dataset of 729 females].

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No total litter losses by resorption were observed in any of the treated groups.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of early and late resorptions was unaffected by treatment with the test item up to 200 mg/kg bw/day.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses was unaffected by treatment with the test item up to 200 mg/kg bw/day.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related difference was observed in pregnancy duration between the control and treatment groups up to and including 200 mg/kg bw/day. One animal at 80 mg/kg bw/day started to deliver early, but this was considered unrelated to treatment.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No effects in the number of pregnant females were observed. In total, 20, 17, 18 and 20 females surviving until scheduled necropsy were gravid with viable fetuses.
1/21 females in the control group, 3/21 females in the 30 mg/kg bw/day group, 3/21 females in the 30 mg/kg bw/day group and 1/21 females in the 200 mg/kg bw/day group were not pregnant.

Summarized results 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:

systemic

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effect was observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weights of test material-treated females were slightly lower compared to concurrent control, without reaching statistical significance (up to -8.31% lower for combined fetal weights at 200 mg/kg bw/day). This was considered a treatment-related but minor effect and secondary to the systemic maternal toxicity. It was therefore not considered adverse.

Mean combined (male and female) fetal body weights were 41.25, 39.15, 38.91 and 37.82 g for the control, 30, 80 and 200 mg/kg bw/day groups, respectively.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live fetuses was unaffected by treatment with the test item up to 200 mg/kg bw/day.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected at all tested dose levels.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The litter sizes and weights were considered unaffected at all tested dose levels.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external malformations, variations and incidental findings were noted that were considered related to treatment with the test material.
One external malformation was observed. One fetus from the mid-dose group (80 mg/kg bw/day) presented with a hyperflexed forepaw. The single incidence of this malformation rules out a relationship with the test material.
No external variations and incidental findings were observed in any of the fetuses.


Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No skeletal malformations and variations were noted that were considered related to treatment with the test material.
Skeletal malformations were observed in 4 (3), 0 (0), 2 (2), 1 (1) and fetuses (litters) of the control, 30, 80, 200 mg/kg bw/day groups, respectively. The affected structures included the skull, rib, sternebra and vertebra. All skeletal malformations in this study were either only observed in the control group or in individual animals that received test material. Therefore, these malformations were considered not to be related to the treatment with the test material.
With regard to skeletal variations, there was one noteworthy finding in the low-dose group (30 mg/kg bw/day): A statistically significantly increased incidence of unossified pubis was observed when compared to the control group. In each of these fetuses, other bones were observed incompletely ossified or unossified. This indicates that the finding was attributed to the below average fetal weights, rather than as a direct response to the test material. This is further supported by the lack of a dose response relationship.
All other skeletal variations were either scored infrequently, limited to control fetuses, in instances comparable to the control group, and/or not statistically relevant. Therefore, they were also considered not to be associated with exposure to the test material.

Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No visceral malformations, variations and incidental findings were noted that were considered related to treatment with the test material.
Visceral malformations were observed in 4 (3), 1 (1), 3 (3) and 2 (2) fetuses (litters) of the control, 30, 80, 200 mg/kg bw/day groups, respectively. Malformations affected the eye, heart, lung, spleen, subclavian artery, kidney and brain. With the exception of retroesophageal right subclavian artery and malpositioned kidney, all malformations were observed only once in test material groups or were limited to a control fetus, ruling out a relationship with the test material. Both abovementioned exceptions were only observed in one fetus each at 80 and 200 mg/kg bw/day. In the absence of a dose-related incidence, a relationship to treatment with the test material can be excluded.
Visceral variations were observed in the gallbladder, gonads, innominate arteries, liver, lung, spleen, and ureter and were scored either infrequently, occurred within normal biological variation, in the absence of a dose-related trend and/or in instances comparable to the control group and were therefore considered not to be linked to treatment with the test material.
Incidental findings included discoloration of the thymus (one fetus as 80 mg/kg bw/day) and liver lobe (one control fetus). Based on the single incidence and/or as it concerned a finding in a control fetus, these incidental findings were considered not related to treatment with the test material.


Summarized results can be found in Attachment 2 under "Overall remarks, attachments".

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

200 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.

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicicty

Effect level:

200 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.

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). Time-mated female New Zealand White rabbits were treated with the test substance once daily by oral gavage for Days 7-28 post-coitum. Clinical signs were observed at 80 and 200 mg/kg bw/day but were only considered adverse at 200 mg/kg bw/day and treatment related mortality was observed at 200 mg/kg bw/day. Therefore, the NOAEL for the test substance regarding maternal systemic toxicity was set at 80 mg/kg bw/day. No effects were noted regarding developmental toxicity or teratogenicity. Therefore, the NOAEL for developmental toxicity and teratogenicity was set at 200 mg/kg bw/day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 Nov 2021 - 02 Feb 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

Main OECD 414 study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

This guidelines was not fully applicable since the study was intended for dose selection purposes only. Therefore, various examinations were not performed (e.g. soft tissue and skeletal malformations, head examination and anogenital distances of fetuses).

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18 - 20 weeks
- Weight at study initiation: 2930 – 4072 g
- Fasting period before study: not applicable
- Housing: individually housed in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm).
- Diet: pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Switzerland). Animals received 25 g pelleted diet on the day of arrival and 140-160 g pellets on subsequent days. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and fresh vegetables were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): means were 18 - 19
- Humidity (%): 46 to 75
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

The humidity values were outside the targeted range for 1 day with a maximum of 75% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.

IN-LIFE DATES: From: 11 Nov 2021 To: 9 Dec 2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared at least weekly. 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.

VEHICLE
- Concentration in vehicle: 133.3 mg/mL (200 mg/kg bw/day), 200 mg/mL (300 mg/kg bw/day), 266.7 mg/mL (400 mg/kg bw/day)
- Amount of vehicle: 1.5 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item under test conditions were already demonstrated in a previous study from the laboratory (analytical method development and validation study, Test Facility
Study No. 20298606).

Details on mating procedure:

The animals were mated by the supplier one day before arrival at the testing facility.

Duration of treatment / exposure:

Day 7 to Day 28 post-coitum

Frequency of treatment:

once daily, 7 days/week

Duration of test:

until necropsy at Day 29 post-coitum

Dose / conc.:

200 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

400 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in consultation with the sponsor, based on the results of a tolerability study with the test substance in rabbits by oral gavage (Test Facility Study No. 20298602). In this tolerability study, dose levels of 300, 600 and 450 mg/kg bw/day were tested for up to 7 days of treatment. At 600 mg/kg bw/day, one animal was euthanized for humane reasons on Day 7 prior to dosing because of severe body weight loss (10%). All animals at this dose level showed decreased activity and/or erected fur on several occasions from Day 3 onwards. In addition, 2/3 animals showed increased respiratory rate, panting, dilated pupils, prostrate and/or uncoordinated movements on several occasions between Days 3 and 7. Body weight loss was noted in all animals, as well as reduced food consumption. At 450 mg/kg bw/day, all animals were euthanized on Day 6 of treatment for humane reasons as the animals appeared to have some type of episode. All animals showed severe clinical signs including increased respiratory rate, dilated pupils, prostrate, erected fur, uncoordinated movements, decreased activity, lying on side and/or head tilted. In addition, body weight loss (2/3 animals) and reduced food consumption (3/3 animals) was noted. 300 mg/kg bw/day appeared to be tolerated by the animals.

Maternal examinations:

CHECK FOR MORTALITY: Yes
- Time schedule: twice daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily post-dose, starting on Day 7 post-coitum onwards up to and including the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 3, 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum, calculated over each sequent interval and over the total dosing period. Corrected body weight gain will be determined based on Day 7 and Day 29 post-coitum body weights and the gravid uterus weight.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 post-coitum by intravenous injection of sodium pentobarbital.
- Examinations: all animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) will be subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

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:

No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: No
- Fetal body weights: Yes

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences were 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, Fisher's exact test.

Historical control data:

Not provided

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, 1/6 animals was noted with some type of episode (i.e. increased
respiratory rate, prostrate, severely uncoordinated movements, extensor rigidity of both hindlimbs and/or decreased activity) on Day 11 post-coitum, 1 h post-dose. In addition, uncoordinated movements (up to severe) and decreased activity were noted in 6/6 animals from Day 8 post-coitum onwards on several consecutive days of the treatment period. Moreover, lying on side, erected fur and/or eyes partially closed were noted incidentally. Red liquid (up to moderate amount) was present on the manure tray of one animal of this dose group on Days 22, 23 and 27 post-coitum.

In the 300 mg/kg bw/day group, 1/6 animals was noted with some type of episode (i.e. increased respiratory rate, constricted pupils, prostrate, severely uncoordinated movements, extensor rigidity of both hindlimbs and/or decreased activity) on Days 19 and 20 post-coitum, 1 h post-dose. In addition, decreased activity and/or uncoordinated movements (up to severe) were noted in up to 6/6 animals from Day 9 post-coitum onwards on several days of the treatment period. Moreover, prostrate and/or lying on side were noted incidentally.

In the 400 mg/kg bw/day group, 2/6 animals were noted with some type of episode (i.e. increased respiratory rate, prostrate, severely uncoordinated movements, extensor rigidity of both hindlimbs, lying on side and/or decreased activity) on Days 12, 15, 16 and/or 18 post-coitum, 1 h post-dose. In addition, uncoordinated movements (up to moderate) and decreased activity was noted in 6/6 animals on several days of the treatment period, from Day 9 post-coitum onwards. Moreover, prostrate was noted incidentally.

The clinical signs of animals sacrificed in extremis are discussed under "Mortality".

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable

Mortality:

mortality observed, treatment-related

Description (incidence):

1/6 animals of the 200 mg/kg bw/day group, 6/6 animals of the 300 mg/kg bw/day group and 6/6 animals of the 400 mg/kg bw/day group were sacrificed before the end of the study. All these deaths were considered test-item related; 4 animals were sacrificed in extremis and 9 animals were sacrificed as the remainder of the group was sacrificed in extremis and sufficient data was obtained for this dose range finding study. With the exception of the animal of the 200 mg/kg bw/day group and one animal of the 300 mg/kg bw/day group, all animals sacrificed before termination of the study were pregnant with normal implantations (< Day 22 post-coitum) or fetuses (≥ Day 22 post-coitum) for the duration of pregnancy. All animals sacrificed prematurely showed episodes of clinical signs such as head tilt, increased or decreased respiratory rate, shallow breathing, hunched posture, both eyes completely closed, prostrate, severely uncoordinated movements, lying on side, decreased activity, extensor rigidity of both hindlimbs and/or lack of/impaired righting reflex on multiple days, so that it was decided to sacrifice them.

The animal of the 200 mg/kg bw/day group was sacrificed on Day 25 post-coitum. Stable body weight was noted for this animal and its food consumption was considered within normal range during the treatment period. At necropsy, no abnormalities were recorded for this animal.

One animal of the 300 mg/kg bw/day group was sacrificed in extremis on Day 22 post-coitum. Stable body weight was noted for this animal. Its food consumption was reduced from Day 8 post-coitum onwards, up to days with absent food consumption. At necropsy, clear watery cyst on the right oviduct was noted, which is a common finding in rabbits of this strain and age. As all other animals of this dose group presented with similar or more severe clinical signs and based on the presence of similar clinical signs at 200 mg/kg bw/day, remaining animals of this dose group were sacrificed as well on Day 22 post-coitum.

In the 400 mg/kg bw/day group, two animals were sacrificed in extremis on Day 20 post-coitum. Both animals had stable body weights and normal food consumption. No abnormal findings were noted at necropsy. As all other animals at this dose group presented with similar clinical signs of toxicity as these 2 animals, the high dose level was considered too high for dosing in the Main study and the 4 remaining animals in this dose group were sacrificed as well on Day 20 post-coitum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, body weight and body weight gain adjusted for gravid uterus weight was considered unaffected by treatment with the test item.
In the 300 mg/kg bw/day group, slight body weight loss was observed in 3/6 animals between Day 9 - 12 post-coitum and for 2/6 animals between Day 12 - 15 post-coitum. Body weight gain throughout the remainder of the treatment period was in the same range as controls. Body weight gain adjusted for gravid uterus weight could not be determined.
In the 400 mg/kg bw/day group, body weight was stable. Body weight gain adjusted for gravid uterus weight could not be determined.

Summarized results can be found in Attachment 1 (Figures) and 2 (Tables) under "overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption was observed from 200 mg/kg bw/day onwards, based on severity as well as on timing of onset, reaching statistical significance between Day 12 - 15 post-coitum at 300 mg/kg bw/day and Day 15 - 18 post-coitum at 300 and 400 mg/kg bw/day. From Day 24 post-coitum onwards, food consumption at 200 mg/kg bw/day was comparable to control levels.

Summarized results can be found in Attachment 1 (Figures) and 2 (Tables) under "overall remarks, attachments".

Food efficiency:

not examined

Description (incidence and severity):

not applicable

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

not examined

Description (incidence and severity):

not applicable

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:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

not examined

Description (incidence and severity):

not applicable

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 200 and 400 mg/kg bw/day group, macroscopic observations at necropsy did not reveal any alterations that were considered related to treatment with the test item.
In one animal of the 300 mg/kg bw/day group, the colon was distended with gas. Clear watery content was noted in the pericardial space of the heart of an additional animal of this dose group, as well as pale white foci on the heart.

Clear watery cysts on the oviduct and/or ectopic or misshapen splenic tissue were found within all treatment groups including control and were within the range of background findings to be expected for rabbits of this age and strain. Any other remaining findings at necropsy were considered not toxicologically relevant.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not applicable

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 were reported.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Pre- and post-implantation loss was increased in the 200 mg/kg bw/day group (11.2% pre-implantation loss vs 5.22% in the control group, 8.64% post-implantation loss vs 5.28% in the control group). No data was available for animals of the 300 and 400 mg/kg bw/day groups, as they were sacrificed before scheduled death.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Suggest changing to say "No total litter loss by resorption was observed in the control or treated animals".

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Due to the higher pre-implantation loss in the 200 mg/kg bw/day group, higher number of early resorptions compared to the control group (0.8 vs. 0.2 in controls) were noted. No data was available for animals of the 300 and 400 mg/kg bw/day groups, as they were sacrificed before scheduled death.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dead fetuses in the 200 mg/kg bw/day group was considered normal, no dead fetuses were observed in this dose group.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No difference regarding the pregnancy duration was observed between the control and treatment groups.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No difference regarding the number of pregnant animals was observed between the control and treatment groups. 1 female of the 200 mg/kg bw/day group was not pregnant. In the 300 and 400 mg/kg bw/day groups, all but one female (300 mg/kg bw/day) were pregnant with viable fetuses (300 mg/kg bw/day) or normal implantation sites for the duration of gestation (Day 20 post-coitum, 400 mg/kg bw/day). As dosing started on Day 7 post-coitum, the effects seen (i.e. two non-pregnant animals) were not test item related.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In the 200 mg/kg bw/day group, the number of implantations was lower compared to the control group (8.6 vs. 10.5 in controls) as well as the number of corpora lutea (9.6 vs. 11.0 in controls).

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

early or late resorptions

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The weight of fetuses in the 200 mg/kg bw/day group was considered normal. No data was available for animals of the 300 and 400 mg/kg bw/day groups, as they were sacrificed before scheduled death.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

The number of live fetuses in the 200 mg/kg bw/day group was lower compared to the control group (7.8 vs. 10.0 in controls). However, this was considered not a primary effect but secondary due to maternal toxicity. No data was available for animals of the 300 and 400 mg/kg bw/day groups, as they were sacrificed before scheduled death.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Changes in sex ratio:

not examined

Description (incidence and severity):

not applicable

Changes in litter size and weights:

not examined

Description (incidence and severity):

not applicable

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

not applicable

Changes in postnatal survival:

not examined

Description (incidence and severity):

not applicable

External malformations:

no effects observed

Description (incidence and severity):

External examination of the fetuses did not show any abnormalities in the 200 mg/kg bw/day group. No data was available for animals of the 300 and 400 mg/kg bw/day groups, as they were sacrificed before scheduled death.

Summarized results can be found in Attachment 2 under "overall remarks, attachments".

Skeletal malformations:

not examined

Description (incidence and severity):

not applicable

Visceral malformations:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental effects were observed at this dose level.

Remarks on result:

other: The decreased numbers of live fetuses were considered to be secondary due to maternal toxicity. Higher doses could not be evaluated due to severe maternal toxicity.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The present non-GLP dose range finding study was conducted in a similar manner to OECD 414 (2018). Time-mated female New Zealand White rabbits were treated with the test substance once daily by oral gavage for Days 7 - 28 post-coitum.
Severe clinical signs were observed in the mid and high-dose group (300 and 400 mg/kg bw/day), therefore, all animals of these groups were sacrificed prematurely. In the 200 mg/kg bw/day group, episodes similar to the ones seen in higher dose groups were noted, but the majority of the animals survived until scheduled necropsy at Day 29 post-coitum. Since all animals of the 300 and 400 mg/kg bw/day group were sacrificed prematurely, the majority of developmental endpoints could not be evaluated. At 200 mg/kg bw/day group, the number of early resorptions was increased compared to the control group.
In conclusion, no NOAEL for maternal systemic and developmental toxicity could be established. The LOAEL was set at 200 mg/kg bw/day based on the adverse clinical signs seen in all treated groups and the early resorptions seen at 200 mg/kg bw/day.
No significant effects were noted regarding developmental toxicity off the offspring. The only effect seen was a decrease in the number of live fetuses in the 200 mg/kg bw/day group, but this can be attributed to maternal toxicity. Therefore, the developmental NOAEL was set at 200 mg/kg bw/day. The higher doses group could not be evaluated due to the severe maternal toxicity.
Based on the results of this Dose Range Finder, selected dose levels for the Main study (Test Facility Study No. 20298605) were 30, 80 and 200 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies performed with member substances of the Alcohol Ethoxylates (AE) category including data on the target substance. The selected studies are 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, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Studies with alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3)

As briefly described under 'Additional information' in the section 'Effects on fertility' above, the reproductive and developmental toxicity of alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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, 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 93, 89, 93 and 93% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. It is thus considered not affected by treatment. Litter sizes were 12.2, 11.4, 12.2, 10.6 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The slightly lower number of pups/litter recorded in the high dose group was considered related to the lower number of implantation sites in this group. As the difference is slight, this was considered not to be toxicologically relevant. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100, 100, 98 and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Two pups at 300 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 remained within the range considered normal for pups of this age. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not affected by treatment. Viability indices were 100, 98, 100, 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Two pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were missing on PND 3 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose related trend and remained within the range considered normal for pups of this age. 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, 100, 100, 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

One pup at 1000 mg/kg bw/day was missing on PND 7. This pup was most likely cannibalised. No toxicological relevance was attributed to this missing pup since the mortality incidence remained within the range considered normal for pups of this age. Most pups (7/8) of litter No. 80 were dehydrated on PND 7, as this occurred on a single day only, this was considered not to be toxicologically relevant. No clinical signs were noted in other litters at 1000 mg/kg bw/day or at other dose levels. A slightly lower body weight gain was noted for pups at 1000 mg/kg bw/day, (-10% for males and females combined on PND 13; not statistically significant). Based on the magnitude of the change and as the body weights of other litters were in the range of control litters, this was considered not to be toxicologically relevant. Sex ratio and anogenital distance (absolute and normalized for body weight) in male and female pups were considered not affected by treatment. A statistically significantly higher mean corrected anogenital distance for female pups at 1000 mg/kg bw/day was considered not to be toxicologically relevant in absence of a dose related trend. 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 of PND 14-16 pups were increased (1.23x and 1.22x of control for males and females, respectively) at 1000 mg/kg bw/day. Serum T4 levels of PND 14-16 pups at 100 and 300 mg/kg bw/day remained in the same range as controls. No test item-related effect was noted for serum TSH levels of PND 14 -16 pups or serum T4 levels in PND 4 pups. No macroscopic findings were noted. No indication of a teratogenic effect of the test item was observed.

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

Alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) was administered to female Wistar (Crl: WI(Han) rats in a Prenatal developmental toxicity study according to OECD guideline 414 under GLP conditions (Sasol, 2022b). Groups of 22 dams were administered doses of 100, 300 and 1000 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.

Based on 14/21 early deliveries in subgroup 2, along with high fetal weights for this subgroup, and low fetal weights for subgroup 1, it is suspected that subgroups 1 and 2 may have been switched after mating. This would indicate that animals of subgroup 1 (female Nos. 1-6, 23-27, 45-49, 67-72) were dosed on gestation day 5-19 and sacrificed on gestation day 20, and animals of subgroup 2 (female Nos. 7-11, 28-33, 50-55, 73-77) were dosed on gestation day 7-21 and sacrificed on gestation day 22. Subgroups 3 and 4 were dosed according to schedule (i.e., dosing on gestation day 6-20 with sacrifice on gestation day 21). 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 substance-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.

Salivation was seen on several occasions following dosing in 5/22 mid-dose females and 9/22 high-dose females, respectively. The salivation was not considered toxicologically relevant and was considered a physiological response related to the gavage procedure rather than a sign of systemic toxicity. A hunched posture was observed on several occasions during gestation day 7-21 in 1/22 mid-dose females and 2/22 high-dose females, respectively. In the high-dose group, erected fur was noted for 4/22 females on several occasions and 1/22 females had abnormal breathing sounds on gestation day 16. These signs were considered treatment-related but not adverse.

The body weight was evaluated by subgroup, due to the suspected switch of subgroups 1 and 2 after mating.

In the high-dose group, all subgroups showed toxicologically relevant differences to the control. The body weight gain for subgroups 3+4 (subgroup 3 and 4 combined) was lower than control throughout the dosing period (gestation day 6 – 20), with a total mean body weight gain of 37% vs 48% in concurrent control. A similar effect on body weight gain was observed in subgroup 1 (35% vs 47% in the control) and in subgroup 2 (22% vs 37% in concurrent control). For subgroup 2, the body weight of Day 18 post-coitum was used as the last time point for evaluation as several animals of this subgroup delivered early. The terminal body weight was 11%, 7% and 7% lower than the control for subgroup 1, 3 and 4, respectively. In subgroup 2, the mean body weight of animals that delivered early was 4% lower than control at the end of the dosing period. For the two females in subgroup 2 that did not deliver early, the body weight was 9% lower than concurrent control on gestation day 21. Four (4/22) high-dose females showed a slight body weight loss (<5%) between gestation day 6 and 9, but gained weight during all other intervals before and after this one. The mean terminal body weight was significantly lower in high-dose females than in control females. The effect on body weight gain in high-dose females in considered to be treatment-related and adverse. In high-dose females the body weight gain corrected for gravid uterus weight was slightly lower (5 - 15%) compared with control, although not significantly in any subgroup. The mid-dose females showed a lower body weight gain (not significant) compared with control in subgroup 1 only, and this is not considered to be toxicologically relevant.

In the mid-dose group the food intake for Subgroup 1 animals was slightly reduced during the complete dosing period. The mean overall food intake was -11% compared to the control, but the difference did not reach statistical significance. Because a slightly change was only noted in one subgroup this finding was not considered adverse in the mid-dose group. In the high-dose group, the food consumption was reduced in all subgroups during the complete dosing period. For most of the intervals in Subgroup 1 and 3+4, these differences reached statistical significance. In Subgroup 2, food consumption was also decreased in all intervals with the exception of gestation day 18 - 21, but none of the differences reached statistical significance. For Subgroups 1, 2 and 3+4, the mean overall food intake was 24, 11 and 17% lower than concurrent controls, respectively. For Subgroup 1 and 3+4, this difference was statistically significant. The reduced food consumption is considered to be toxicologically relevant.

Triiodothyronine (T3) levels were statistically significantly decreased in mid- and high-dose females (0.86x and 0.79x, respectively), compared with control. In addition, thyroid stimulating hormone (TSH) levels were increased in mid- and high-dose females (1.31x and 1.66x, respectively) compared with control. The difference in TSH level reached statistical significance only for the high-dose group. As all the mean values fell within the available historical control data range they were considered non-adverse.

High-dose females had lower absolute thyroid gland weight (16% lower than control, significant) and relative thyroid gland weight (10% lower, non-significant). This difference was considered non-adverse due to the small difference and the lack of microscopic correlate. A pale discoloration of the liver was noted in 3 females (2/22 in the mid-dose group, 1/22 in the high-dose group group). The liver of the female in the high-dose group was also enlarged. 1/22 high-dose females had a thick, non-glandular stomach. Given the incidental nature of these findings, no association with histopathology and/or the absence of a dose-related response, these findings were considered unrelated to treatment with the test substance.

Three of twenty-one (3/21), 5/21, 4/21 and 2/22 females in the control, low-, mid- and high-dose groups, respectively, delivered early. All early deliveries were by females in subgroup 2. Subgroups 1 and 2 may have been switched after mating and subgroup 2 was sacrificed on gestation day 22 instead of 21, as planned. Considering the average gestation period, deliveries were to be expected from gestation day 22 and this is not a treatment-related effect.

No treatment-related effects were noted on mortality / moribundity, 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.

The maternal NOAEL systemic was set at 300 mg/kg bw/day based on the reduced body weight gain and reduced food consumption at the highest dose level of 1000 mg/kg bw/day.

In the offspring, the mean body weight varied by subgroup. The fetal body weight for all dose groups of subgroup 1 (including control) were below the lower range of the available historical control data. In contrast, fetal body weight for all dose groups of subgroup 2 (including control) were above the upper range of the available historical control data. The fetal body weight of the control group were within range of the available historical control data. These variations were attributed to the suspected switch of subgroup 1 and 2, as fetuses were likely weighed on gestation day 20 in subgroup 1 and on gestation day 22 in subgroup 2. In the high-dose group, mean fetal body weights in subgroup 1 and 3+4 (male, female and combined) were lower than for control (combined body weights 8% and 4% lower than concurrent control in subgroup 1 and 3+4, respectively; not statistically significant). The mean fetal body weights of subgroup 4 were below or at the lower end of the historical control range. For subgroup 2, mean fetal body weights (male, female and combined) were in the same range as the concurrent control.

No external malformations or variations were observed. There were no treatment-related visceral malformations or variations, nor any skeletal malformations or variations. No treatment-related effects were noted on litter size, sex ratio and anogenital distance.

Based on reduced offspring body weight at 1000 mg/kg bw/day, the NOAEL for developmental toxicity is 300 mg/kg bw/day. The NOAEL teratogenicity is 1000 mg/kg bw/day.

Alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) was administered to female New Zealand White rabbits in a Prenatal developmental toxicity study according to OECD guideline 414 under GLP conditions (Sasol, 2022c). Groups of 22 dams were administered doses of 30, 80 and 200 mg/kg bw/day by oral gavage, 7 days a week on gestation day 7 - 28. 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, head examination and skeletal examination.

A total of 1/22, 2/22, 1/22 and 1/22 females in the control, low- mid- and high-dose group, respectively, were sacrificed prematurely. The animal in the control group was sacrificed in extremis for animal welfare reasons on gestation day 17 as it had limited to no food consumption for 7 consecutive days. Slight body weight loss was noted for this animal on gestation day15 (3% compared to the highest recorded body weight on site). At necropsy, ectopic splenic tissue and clear watery cysts on the oviducts were noted, which are findings occasionally seen among rabbits used in this type of study. This animal was pregnant with normal implantation sites for the duration of pregnancy.

One of the 2 animals in the low-dose group was sacrificed in extremis on gestation day 8as it did not acclimatize to the facility properly and showed (almost) absent food consumption. Between Days 3 (first body weight recorded on site) and 7 (start dosing) the animal lost 4% of its body weight. The animal was noted with some type of abnormality on its left eyelid, causing a swollen eyelid, red discoloration of the eyeball and clear as well as mucoid discharge from the eye. At necropsy, a white nodule with hair on the sclera of the left eye was observed, as well as prominent lobular architecture of the liver. This animal was pregnant with normal implantation sites for the duration of pregnancy. The second animal of this dose group sacrificed in extremis (gestation day 21)had low to no food consumption for 8 consecutive days. In addition, moderate body weight loss was noted for this animal (6% compared to the highest recorded body weight on site). At necropsy, prominent lobular architecture of the liver was noted. This animal was pregnant with normal implantation sites for the duration of pregnancy.

The animal in the mid-dose group was sacrificed early as it started to deliver its litter early on gestation day 24. On gestation day 23, erected fur and decreased activity were noted. Except for slight body weight loss during gestation day 12 - 15, normal body weight gain was noted for this animal. It had a stable food consumption with the exception of the 2 days prior to its delivery, during which low to absent food consumption was noted. At necropsy, no macroscopic abnormalities were noted. This female had 3 dead fetuses.

In the high-dose group, one female was sacrificed in extremis on gestation day 17 as it presented with a behavioural episode on gestation day 15, 16 and 17. These episodes consisted of the following clinical signs: increased respiratory rate, severely uncoordinated movements, lying on its side, head tilt, prostrate position, hunched posture, decreased activity and/or extensor rigidity of both hind limbs. The body weight (gain) for this animal was considered within the expected range. Average food consumption was noted for this animal up to gestation day 12 and lower food consumption from gestation day 12 onwards. At necropsy, ectopic splenic tissue was noted, which is a finding that is occasionally seen among rabbits used in these types of study and was considered to have no toxicological significance. This animal was pregnant with normal implantation sites for the duration of pregnancy. The early sacrifice of the high-dose animal was considered treatment-related due to the severe clinical signs. The other 4 early sacrifices in the control, low- and mid-dose groups were considered incidental as the reasons for the unscheduled sacrifice were varied and not considered to be caused by the test substance.

In the mid-dose group, decreased activity was noted in 9/22 animals on individual or multiple days during gestation day21 - 28. 1/22 females was observed with a thin appearance from gestation day26 to 28 and 1/22 showed uncoordinated movement on gestation day 26 - 28.

In the high-dose group, behavioural episodes were noted on several (consecutive) days of treatment from gestation day 8 onwards. Uncoordinated movement was noted in 19/22 females on several occasions on gestation day 8 – 28, while 16/22 females were observed lying on their side on multiple occasions from gestation day 10 onwards. Decreased activity was observed in 22/22 animals from gestation day 8 onwards, on multiple occasions. In at least 2/22 animals (including the animal that was sacrificed early) one or several clinical signs were noted on one or a few occasions: abnormal respiratory rate, shallow breathing, erected fur, hunched posture and/or prostrate position. The clinical signs were considered toxicologically relevant in the high-dose group.

In the low- and mid-dose group the overall mean body weight gain (gestation day 7 - 29) was decreased by 9 and 11%, respectively, compared to the control group (not statistically significant). In the high-dose group the overall mean body weight gain (gestation day 7 - 29) was decreased by 12% compared to the control group (not statistically significant). As the overall change was minimal for all groups and the intervals (e.g. gestation day 7 – 9) showed both a higher and a lower body weight gain than the control for all treatment groups, this was not considered an adverse effect. A mean body weight loss was observed for all groups, including control, when adjusting the total body weight changes for the gravid uterus weight (-91.8, -117.4, -141.8 and -151.2 g in the females in the control, low- mid- and high-dose group, respectively). The weight changes were similar between all groups and were not considered to be treatment-related.

The mean food consumption for gestation day7 – 29 was reduced by 3 - 7% in all dose groups, compared with the control. None of the variations reached statistical significance and they were not considered to be adverse.

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, 2/21 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. Among the females that were sacrificed unscheduled 2/2 showed prominent lobular architecture of the liver. The liver changes were considered treatment-related, 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.

The maternal NOAEL systemic was set at 80 mg/kg bw/day.

In the offspring, the mean body weight for male and female fetuses combined was 5, 6 and 8% lower in thein 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 treatment-related effects were noted on litter size and sex ratio. There were no treatment-related external, skeletal or visceral malformations or variations.

The NOAEL developmental is 200 mg/kg bw/day and the NOAEL teratogenicity is 200 mg/kg bw/day.

 

A dose range-finder study in pregnant rabbits was performed with Alcohols, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3)(20298604, Sasol, 2022d). In this study, 6 pregnant rabbits/group were exposed to the test substance via oral gavage at dose levels of 200, 300, and 400 mg/kg bw/day,7 days a week on gestation day 7 - 28. In total, 13 animals (6 at 400 mg/kg bw/day, 6 at 300 mg/kg bw/day and 1 at 200 mg/kg bw/day) did not survive until scheduled necropsy. All these deaths were considered test-substance related; 4 animals (1/6, 1/6 and 2/6 at 200, 300 and 400 mg/kg bw/day, respectively) were sacrificed in extremis, based on severe clinical signs of toxicity, highly suspected to be of neurological origin. These signs included: increased respiratory rate, shallow breathing, prostrate, hunched posture, both eyes completely or partially closed, severely uncoordinated, lying on side, extensor rigidity of both hindlimbs, decreased activity, head tilt and/or lack of/impaired righting reflex. In addition, 9 animals (5/6 and 4/6 at 300 and 400 mg/kg bw/day, respectively) were sacrificed on veterinary advice as these animals presented with similar clinical signs of toxicity. Since the remainder of the group was sacrificed in extremis, sufficient data was obtained to consider 300 and 400 mg/kg bw/day too high for dosing in the main study. Moreover, lower food consumption was observed from 200 mg/kg bw/day, based on the size of the effect as well on timing of onset, with apparent recovery by the end of the treatment period at 200 mg/kg bw/day only. At 300 mg/kg bw/day, slight body weight loss was noted in 3/6 animals gestation day 9-12 and for 2/6 animals gestation day 12-15, followed by stable body weight gain throughout the remainder of the treatment period. Body weight gain at 200 mg/kg bw/day was comparable to controls and considered unaffected by treatment. At 400 mg/kg bw/day body weight was considered stable. With the exception of 1/6 dams in the 200 mg/kg bw/day group and 1/6 dams in the 300 mg/kg bw/day group, all animals were pregnant at (early) termination. As all animals at 300 and 400 mg/kg bw/day were sacrificed prior to gestation day 29, there was no data available to investigate remaining maternal and fetal parameters at these dose levels. At 200 mg/kg bw/day, the number of corpora lutea was lower, as was the number of implantations. Pre- and post-implantation loss was increased, which was caused by a higher number of early resorptions.

Based on the adverse findings and in consultation with the Sponsor, the following dose levels were selected for the main study: 30, 80 and 200 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	80	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, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) is consistent with the overall toxicity to reproduction data for AE substances.

The following NOAELs were set:

Oral (rat, OECD 414): NOAEL (developmental toxicity) = 300 mg/kg bw/day

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

Oral (rabbit, OECD 414): NOAEL (developmental toxicity) = 200 mg/kg bw/day

Oral (rabbit, OECD 414): NOAEL (teratogenicity) = 200 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, C12-14, ethoxylated (CAS No. 68439-50-9, EC No. 500-213-3) 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.