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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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.

 

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Alcohols, C12-14, ethoxylated
EC Number:
500-213-3
EC Name:
Alcohols, C12-14, ethoxylated
Cas Number:
68439-50-9
Molecular formula:
n.a.
IUPAC Name:
Alcohols, C12-14, ethoxylated, < 2.5 EO

Test animals

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.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant
- Age at study initiation: Males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: Males 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
- Acclimation period: 7 days

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

IN-LIFE DATES: From 5 FEB 2020 to 30 MAR 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human
exposure during manufacture, handling or use of the test item.
Vehicle:
corn oil
Details on oral 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
- Batch: MKCH1635 + MKCG3257, MKCK6411
- Specific gravity: 0.92
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 less than or equal to10%.
- 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
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study (Test Facility Reference No. 20218714) with oral gavage administration of Alcohols, C12-14, branched and linear, ethoxylated (2 EO) in rats, and in an attempt to produce graded responses to the test item.

- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid,
gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea, urinary bladder, uterus, vagina.
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.

Results and discussion

Results of examinations

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.
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:
no effects observed
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 ranges, 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:
effects observed, treatment-related
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 higher weights (absolute and/or relative to body weights) were noted in the
male liver and kidney at 300 and 1000 mg/kg bw/day and female adrenal gland at 1000 mg/kg
bw/day as shown in the Table 1.
A higher liver weight (absolute and relative to body weight) was recorded for males at 300
and 1000 mg/kg bw/day. The microscopic correlate at 1000 mg/kg bw/day was centrilobular
hepatocellular hypertrophy.
Higher kidney weights were recorded for males at 300 mg/kg bw/day (absolute) and at
1000 mg/kg bw/day (absolute and relative to body weight). There was no microscopic
correlate for this weight change.
Higher adrenal gland weights were recorded for females at 1000 mg/kg bw/day (absolute and
relative to body weights). The microscopic correlate for this weight change was cortical
hypertrophy, zona fasciculata.
There were no other test item-related organ weight changes. Some organ weight differences
were statistically significant when compared to the control group; a lower absolute prostate
gland weight and higher testes and epididymides weight relative to body weight at 1000
mg/kg bw/day. This was considered to be the result of a test item-related lower final body
weight in males at 1000 mg/kg bw/day. As changes were slight this was considered not
adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
An accentuated lobular pattern in the liver was recorded for a single male at 100 mg/kg
bw/day, two males at 300 mg/kg bw/day and three males at 1000 mg/kg bw/day. At 1000
mg/kg bw/day the microscopic correlate for this finding was centrilobular, hepatocellular
hypertrophy. This finding was interpreted to be non-adverse at the recorded minimal severity
and in absence of any additional inflammatory or degenerative alterations.
The remainder of the recorded macroscopic findings were within the range of background
gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
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

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion