Hexadecan- l-ol, ethoxylated

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jul 2020 - 06 Apr 2021

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:

14 Jul 2020 - 06 Apr 2021

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.

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 299-347 g, females 186-225 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: 6 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 52-76
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From: 14 JUL 2020 to 16 SEP 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.
- The dosing formulations were prepared weekly as a solution through heating to a maximum temperature of 40 ± 1°C under continuous stirring for at least 30 minutes to obtain visual homogeneity.
-Test item dosing formulations were kept at 40 ± 1°C until and during 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 (Test Facility Study No. 20243920) to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Details on mating procedure:

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 (Test Facility Study No. 20243920).
- 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 to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

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 50-64 days.
Females which failed to deliver or had a total litter loss 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 in rats (Test Facility Reference No. 20243922), and in an attempt to produce graded responses to the test item.
F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

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

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

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

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

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations: testis weight, epididymis weight.

Litter observations:

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

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


GROSS EXAMINATION OF DEAD PUPS
Pups found dead were examined for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

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

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

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

Postmortem examinations (offspring):

SACRIFICE
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 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.

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 were used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.

Mating index (%): Number of females mated/Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females/Number of females mated x 100

Offspring viability indices:

For each group, the following calculations were performed. Group mean values of duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group:

Gestation index (%):Number of females with living pups on Day 1/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%):Total number of offspring born/Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering/Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check/
Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related salivation was noted during daily detailed clinical observations starting at 100 mg/kg bw/day in males and at 300 mg/kg bw/day in females in a dose-related manner.
Incidental findings that were noted included alopecia, rales and piloerection. 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.
No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was related to treatment with the test item up to 1000 mg/kg bw/day.
One female from the control group was euthanized in extremis on Day 2, as it had broken its left front paw after jumping out of its cage. As this accidental injury occurred during the replacement period, this female was replaced by an alternate female.
One female at 1000 mg/kg bw/day was scheduled for euthanasia on Lactation Day 2, as it had a total litter loss.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were affected by treatment with the test item at 100, 300 and 1000 mg/kg bw/day with the following statistically significant changes distinguished treated from control animals:
• Mean glucose concentration was increased (1.40x, 1.26x and 1.27x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean cholesterol concentration was increased (1.36x of control) at 1000 mg/kg bw/day in males.
• Mean potassium concentration was increased (1.10x, 1.08x and 1.09x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean bile acid concentration was increased (1.57x and 2.09x of control) at 300 and 1000 mg/kg bw/day in females, respectively.
Mean values, including potassium concentration in treated males and bile acid concentration in females , achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution or corroborative findings in the opposite sex were therefore considered to be non-adverse and of no toxicological significance.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item due to the minimal magnitude of the change and/or absence of a dose-related response

Endocrine findings:

not specified

Description (incidence and severity):

Mean serum levels of total T4 was statistically significantly decreased at 1000 mg/kg bw/day in F0 males, but not in F0 females.
Mean serum levels of total TSH was statistically significantly increased at 300 mg/kg bw/day in F0 males. In the absence of a dose-related trend, this change was considered to be unrelated to treatment with the test item.
The increase in mean serum levels of TSH in F0 females at 300 and 1000 mg/kg bw/day was considered a result of one or two outliers and was therefore considered to be of no toxicological relevance. In the absence of a dose-related trend, the increase at 100 mg/kg/day was considered to be of no toxicological relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment up to 1000 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength and motor activity was similar between control and high dose animals.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the non-glandular stomach and pituitary gland at 1000 mg/kg bw/day in males and in the jejunum at 300 and 1000 mg/kg bw/day in both sexes. These findings are summarized in Table 3 and Table 4.

Non-glandular stomach (forestomach): Squamous cell hyperplasia of the non-glandular epithelium was observed at a minimal degree in 3/5 males at 1000 mg/kg bw/day. This finding was in two males accompanied by (sub)mucosal (lympho)granulocytic infiltrate, and in one of the two also by a slight ulcer of the epithelium. These findings were considered adverse.

Pituitary gland: an increased incidence and severity of hypertrophy and vacuolation of cells of the pars distalis was observed at 1000 mg/kg bw/day in 3/5 males (up to slight degree). These were considered to be non-adverse test item-related morphologic alterations.

Jejunum: Lymphangiectasis (i.e. dilated lacteals in the villi) was noted in 1/5 males and 3/5 females at 300 mg/kg bw/day (up to a slight degree) and in 4/5 males and 5/5 females at 1000 mg/kg bw/day (up to moderate degree). These were considered to be non-adverse test item-related morphologic alterations.

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.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days. An acyclic cycle was noted for a control female and a female receiving 300 mg/kg bw/day during the premating period, which both littered normally. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stage dependent qualitative evaluation of spermatogenesis in 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):

Mating index was not affected by treatment with the test item up to 1000 mg/kg bw/day. All paired females showed evidence of mating, which resulted in a mating index of 100% for all groups.
Precoital time was not affected by treatment with the test item up to 1000 mg/kg bw/day. All paired females showed evidence of mating within 6 days, except for one female receiving 300 mg/kg bw/day for which mating took 14 days. Given the single occurrence and in the absence of a dose-related trend, this longer mating period was considered not related to treatment with the test item.
Number of implantation sites was not affected by treatment with the test item up to 1000 mg/kg bw/day.
The mean number of implantation sites were 12.0, 12.3, 11.6 and 11.4 for the control, 100, 300 and 1000 mg/kg bw/day groups.
Fertility index was not affected by treatment with the test item up to 1000 mg/kg bw/day.
The fertility indices were 90, 100, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One control female was not pregnant and was therefore not related to treatment with the test item.
Gestation index and duration of gestation were considered not to be affected by treatment
with the test item up to 1000 mg/kg/day.
The gestation indices were 100, 90, 100 and 100% for the control, 100, 300 and
1000 mg/kg/day groups, respectively.
The failed pregnancy of one female at 100 mg/kg/day, without related histopathology
changes in reproductive organs, was judged to be unrelated to treatment due to the incidental
occurrence.
No signs of difficult or prolonged parturition were noted among the pregnant females.

Key result

Dose descriptor:

NOAEL

Remarks:

Local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: based on the microscopic findings in the non glandular stomach (forestomach) in males at 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

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):

No clinical signs occurred among pups that were considered to be related to treatment with the test item up to 1000 mg/kg bw/day.
Clinical signs observed were:
One pup from control group pale on Day 4
One pup at 100 mg/kg bw/day with black tail at apex on first litter check and then missing tail on Day 13
One pup at 300 mg/kg bw/day with blue spot on neck on day of necropsy
One pup at 1000 mg/kg bw/day with wound to front leg on Day 6 was euthanized in extremis
One pup at 1000 mg/kg bw/day dehydrated with no milk in stomach on Day 1

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 1000 mg/kg/day.
The live birth index was 100% for all groups. One pup of the control group, one pup at 100 mg/kg/day, one pup at 300 mg/kg/day and one pup at 1000 mg/kg/day were missing on PND 2 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.
One female at 1000 mg/kg/day had a total litter loss on Lactation Day 2 as it only had one pup which was dehydraded and had no milk in its stomach on PND 1 and went missing on PND 2. At the incidence observed, no toxicological relevance was attributed to the total litter loss.
One pup at 1000 mg/kg/day was euthanized in extremis on PND 6 as it had a wound on its left front leg. No toxicological relevance was attributed to this mortality, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 in male and female 14-16 pups were considered not to be affected by treatment with the test item up to 1000 mg/kg/day.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Mean (normalized) anogenital distance of female pups was decreased at 300 and 1000 mg/kg/day (not always statistically significant) was not considered toxicologically relevant. Mean (normalized) anogenital distance of male pups was not affected with treatment with the test item up to 1000 mg/kg/day.

Nipple retention in male pups:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment up to 1000 mg/kg/day. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically relevant effects observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Data source

Reference

Title:

Unnamed

Year:

2021

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

Test material information

Constituent 1

Reference substance name:

Hexadecan- l-ol, ethoxylated

EC Number:

500-014-1

EC Name:

Hexadecan- l-ol, ethoxylated

Cas Number:

9004-95-9

Molecular formula:

C20H42O3

IUPAC Name:

2-[2-(Hexadecyloxy)ethoxy]ethanol

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.

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 299-347 g, females 186-225 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: 6 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 52-76
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
From: 14 JUL 2020 to 16 SEP 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.
- The dosing formulations were prepared weekly as a solution through heating to a maximum temperature of 40 ± 1°C under continuous stirring for at least 30 minutes to obtain visual homogeneity.
-Test item dosing formulations were kept at 40 ± 1°C until and during 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 (Test Facility Study No. 20243920) to select corn oil as the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 5 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (Test Facility Study No. 20243920).
- 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 to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

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 50-64 days.
Females which failed to deliver or had a total litter loss were treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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 in rats (Test Facility Reference No. 20243922), and in an attempt to produce graded responses to the test item.
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

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.

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 according 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 were 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 was significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related salivation was noted during daily detailed clinical observations starting at 100 mg/kg bw/day in males and at 300 mg/kg bw/day in females in a dose-related manner.
Incidental findings that were noted included alopecia, rales and piloerection. 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.
No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was related to treatment with the test item up to 1000 mg/kg bw/day.
One female from the control group was euthanized in extremis on Day 2, as it had broken its left front paw after jumping out of its cage. As this accidental injury occurred during the replacement period, this female was replaced by an alternate female.
One female at 1000 mg/kg bw/day was scheduled for euthanasia on Lactation Day 2, as it had a total litter loss.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology parameters of treated rats were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Any statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item 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 up to 1000 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were affected by treatment with the test item at 100, 300 and 1000 mg/kg bw/day with the following statistically significant changes distinguished treated from control animals:
• Mean glucose concentration was increased (1.40x, 1.26x and 1.27x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean cholesterol concentration was increased (1.36x of control) at 1000 mg/kg bw/day in males.
• Mean potassium concentration was increased (1.10x, 1.08x and 1.09x of control) at 100, 300 and 1000 mg/kg bw/day in males, respectively.
• Mean bile acid concentration was increased (1.57x and 2.09x of control) at 300 and 1000 mg/kg bw/day in females, respectively.
Mean values, including potassium concentration in treated males and bile acid concentration in females , achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution or corroborative findings in the opposite sex were therefore considered to be non-adverse and of no toxicological significance.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment with the test item due to the minimal magnitude of the change and/or absence of a dose-related response

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean serum levels of total T4 was statistically significantly decreased at 1000 mg/kg bw/day in F0 males, but not in F0 females.
Mean serum levels of total TSH was statistically significantly increased at 300 mg/kg bw/day in F0 males. In the absence of a dose-related trend, this change was considered to be unrelated to treatment with the test item.
The increase in mean serum levels of TSH in F0 females at 300 and 1000 mg/kg bw/day was considered a result of one or two outliers and was therefore considered to be of no toxicological relevance. In the absence of a dose-related trend, the increase at 100 mg/kg/day was considered to be of no toxicological relevance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment up to 1000 mg/kg bw/day.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength and motor activity was similar between control and high dose animals.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A test item-related increase in adrenal gland weights was noted at 1000 mg/kg bw/day in males as shown in Table 1 and possible test item-related increased liver weights were noted at 1000 mg/kg bw/day in females as shown in Table 2. These findings were considered as non-adverse since these changes were not associated with any adverse pathological alterations.
Any other differences, including those that reached statistical significance (relative kidney weights at 1000 mg/kg bw/day in males) were considered not to be related to treatment with the test item due to the direction of the change, lack of dose-related pattern, absence of microscopic correlate and/or general overlap and variability in individual values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the non-glandular stomach and pituitary gland at 1000 mg/kg bw/day in males and in the jejunum at 300 and 1000 mg/kg bw/day in both sexes. These findings are summarized in Table 3 and Table 4.

Non-glandular stomach (forestomach): Squamous cell hyperplasia of the non-glandular epithelium was observed at a minimal degree in 3/5 males at 1000 mg/kg bw/day. This finding was in two males accompanied by (sub)mucosal (lympho)granulocytic infiltrate, and in one of the two also by a slight ulcer of the epithelium. These findings were considered adverse.

Pituitary gland: an increased incidence and severity of hypertrophy and vacuolation of cells of the pars distalis was observed at 1000 mg/kg bw/day in 3/5 males (up to slight degree). These were considered to be non-adverse test item-related morphologic alterations.

Jejunum: Lymphangiectasis (i.e. dilated lacteals in the villi) was noted in 1/5 males and 3/5 females at 300 mg/kg bw/day (up to a slight degree) and in 4/5 males and 5/5 females at 1000 mg/kg bw/day (up to moderate degree). These were considered to be non-adverse test item-related morphologic alterations.

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.

Other effects:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.
The stage dependent qualitative evaluation of spermatogenesis in the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

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

2

Key result

Dose descriptor:

NOAEL

Remarks:

Local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant efects observed

3

Key result

Dose descriptor:

LOAEL

Remarks:

local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no