Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jan 2019 - 15 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jan 2019 - 15 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity/Composition correction factor: No correction factor required (substance is a UVCB)
Species:
rat
Strain:
other: Crl: WI(Han)
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks old (males), 13-14 weeks old (females)
- Weight at study initiation: between 261 and 313 g (males), between 209 and 248 g (females)
- Fasting period before study: no
- Housing: On arrival and following 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 Macrolon plastic cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages), with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. During motor activity measurements, animals had no access to food or water for a maximum of 2 hours. Males were fasted overnight with a maximum of 24 hours before necropsy, females were not fasted.
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 45 to 49%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From 31 Jan 2019 to 8 Apr 2019
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.
The dose levels were selected based the results of a 14-day Dose Range Finder with oral gavage administration of C12-14 Alkyl ethoxylated glycidylether in rats.
Vehicle:
water
Details on oral exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Dose volume: 5 mL/kg. The dose volume for each animal was based on the most recent body weight measurement.
The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (approximately 500 mg) were analysed for concentration and homogeneity. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of target concentration.
Homogeneity 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 and demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 to 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 67 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40 to 43 days.
Two females from the control group and 2 females from the mid dose group were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily
Dose / conc.:
83 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected based on the results of a dose range finder study. Three females were dosed once daily by oral gavage at 500 mg/kg bw/day, and 3 females were dosed at 1000 mg/kg bw/day for 7 days a week for 14 days. The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg bw/day). Mortality was checked twice daily throughout the study. Clinical Observations were done at least daily up to the day prior to necropsy, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights were measured on Day 1 prior to dosing and on Days 4, 8, 11 and 14. Food Consumption was monitored over Days 1-4, 4-8, 8-11 and 11-14. All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy.
Positive control:
Not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals. The peak effect of occurrence of clinical signs occurred directly after dosing. Therefore, clinical observations and functional observation tests were conducted directly after dosing.

BODY WEIGHT: Yes
- 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: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. 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.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells Platelets, Reticulocytes (absolute).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate.

Serum was analyzed for total Thyroxine (T4) for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded

In addition, coagulation parameters were analysed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after completion of clinical observations.
The following tests were performed: Hearing ability, pupillary reflex, static righting reflex.
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal.
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

Sacrifice and pathology:
SACRIFICE
Scheduled euthanasias as follows:
- Males: after completion of mating period, minimum 28 days of administration.
- Females: PND 14-16

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS
- On selected 5 animals/sex/group: according to guidelines.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.

HISTOPATHOLOGY: Yes
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: according to guidelines.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle,and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.
- For the testes of all selected males of the low and high dose groups, and one male of the control group, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Other examinations:
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.
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 to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made: Low dose group vs. Control group; mid dose group vs. Control group; High dose group vs. Control group.
Parametric: 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 was 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.
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations up to 83 mg/kg bw/day (low dose group). No findings were noted during the weekly arena observations in this study. Salivation was seen after dosing among animals of the 250 (mid dose group) and 750 mg/kg bw/day (high dose group) from the first week of the treatment period onwards and was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
One female of the 750 mg/kg bw/day (high dose group) was euthanized on Lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in body weights and body weight gain were observed up to 250 mg/kg bw/day (mid dose group). At Day 8 of pre mating period, body weight gain in males at 750 mg/kg bw/day (high dose group) was decreased to 0.78x when compared with control. During the following weeks of treatment, body weight gains were considered normal. At the end of the post coitum and lactation period, lower (though non-statistically significant) body weights and body weight gains were observed in females at 750 mg/kg bw/day. In absence of statistically significant findings, these changes were considered non adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level up to 250 mg/kg bw/day (mid dose group). In males at 750 mg/kg bw/day (high dose group), absolute and relative food consumption was slightly lower compared with controls (0.92x and 0.96x, respectively) during the first week of treatment. In the following weeks, food consumption levels were considered normal. In females at 750 mg/kg bw/day, absolute food consumption was decreased, when compared with control during the first week of treatment and during the lactation period (0.88x and 0.74-0.76x, respectively), but not during the post coitum period. Similar decreases in relative food consumption were observed during the first 2 weeks of treatment (0.84-0.91x) and the lactation period (0.74-0.77x). Also, relative food consumption levels were decreased during the first three weeks of the post coitum period (0.86-0.91x).
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.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment up to 750 mg/kg bw/day (high dose group). For males, the available data of low and high dose groups indicate that there is no dose related trend and that results are within normal range when compared with the historical control data (For males only one control measurement was available, therefore low and high dose groups data was compared with historical control data). In females, any statistically significant changes noted in hematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters of treated rats were considered unaffected by treatment up to 83 mg/kg bw/day (low dose group). In males, albumin levels were increased with 1.04x and 1.06x at 250 and 750 mg/kg bw/day (mid and high dose groups), respectively, when compared to control, and urea levels were increased with 1.29x at 750 mg/kg bw/day. In females, bile acids levels were increased with 2.88x at 750 mg/kg bw/day. As all values remained within normal range, these changes were considered not toxicologically relevant. Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response.
Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment.
Coagulation parameters: unaffected by treatment up to 750 mg/kg bw/day. For males, the available data of 250 and 750 mg/kg bw/day (mid and high dose groups) indicate that there is no dose related trend and that results are within normal range.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment up to 750 mg/kg bw/day (high dose group). Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval and a decreasing trend in activity over the duration of the test period. All values remained within normal range.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 750 mg/kg bw/day group males (high dose group). There was no microscopic correlate with the increased liver weight. Some organ weight differences were statistically significant when compared to the control group (heart, liver, kidneys in males; relative to body weight only) but were considered to be the result of a test item-related effect on final body weight. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related thickened forestomach was observed in the stomach of 2/10 males treated at 750 mg/kg bw/day (high dose group). The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in one high dose female, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight hyperplasia of the forestomach was recorded in 3 males and 2 females of the 750 mg/kg bw/day group (high dose group). 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.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including highest dose tested (750 mg/kg bw/day)
Key result
Critical effects observed:
no

Results Dose Formulation Analyses:

The concentrations analyzed in the formulations of the low , mid and high dose groups were in agreement with target concentrations (i.e. mean accuracies between 96% and 99%). No test item was detected in the Control group formulation. The formulations of the low dose group and the high dose group were homogeneous (i.e. coefficient of variation ≤ 2.0%).

Results dose range finder study:

No mortality occurred. At 1000 mg/kg bw/day, piloerection was present in all animals during Days 2-8. Hunched posture was present in 2-3/3 animals from Days 3-8 and from Days 11-14. Salivation was observed in 3/3 animals on most days during the second week of treatment. On Day 7, lethargy was observed in 2/3 animals. Body weight was reduced in 1/3 animals during Days 1-4, followed by normal body weights and body weight gain. During Days 1-4, absolute and relative food consumption was reduced. At 500 mg/kg bw/day, a hunched posture was observed in 1/3 animals on Days 13 and 14. Piloerection was seen in 3/3 animals on Day 8. Salivation was observed in 3/3 animals on most days during the second week of treatment. Normal body weights and body weight gain was measured, food consumption was considered within normal range.

At necropsy, no abnormalities were noted at 500 mg/kg bw/day. Kidney weights were within normal range, but relative liver weights were increased with approximately 16% compared with the historical control range. At 1000 mg/kg bw/day, in 1/3 animals the stomach was grown together with the liver and the liver was grown together with the diaphragm. No effects were seen on kidney weights, relative liver weights were increased with approximately 39% compared with the historical control range.

Conclusions:
A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. C12-14 Alkyl ethoxylated glycidylether was administered by daily oral gavage to male and female rats at dose levels of 83, 250 and 750 mg/kg bw/day. Based on the results of this study, the parental NOEL was concluded 250 mg/kg/day (based on lower body weight gain and food consumption, increased liver weights in males, a thickened forestomach in males and hyperplasia in the forestomach in males and females at 750 mg/kg/day). As these fefects were considered to be non-adverse, the NOAEL was found to be 750 mg/kg bw/day.
Executive summary:

C12-14 Alkyl ethoxylated glycidylether has been evaluated in a combined 28-day repeated dose toxicity with reproduction/developmental toxicity screening in rats. The animals were dosed for 29-32 days (males) and average 54 days (females, range 50-67 days and 40-43 days in case non-pregnant or total litter loss).

The applied dose levels of 0, 83, 250, 750 mg/kg/day were based on results of a 14-day range finder involving two groups of 3 female rats at 500 and 1000 mg/kg/day. Effects at 1000 mg involved: hunched posture and lethargy; BW reduction in one animal; reduction absolute and relative food consumption; effects on stomach in one animal at macroscopic examination; increase relative liver weights by 39%.

The following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.  In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).  

Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

The observed decrease in body weight gain in males at 750 mg/kg/day on Day 8, was considered non adverse as body weights were normal and the body weight gain recovered to normal levels in the following weeks. The decrease in body weight gain may have been related to the decreased food consumption levels found during the first week of treatment in males at 750 mg/kg/day.

For females, (relative) food consumption levels were decreased during the premating, post coitum and/or lactation period at 750 mg/kg/day. Though not statistically significant, body weight gains were lower at the end of post coitum and lactation period at 750 mg/kg/day. These findings were likely the result of the decreased litter sizes at 750 mg/kg/day and were considered secondary to developmental and reproductive toxicity.

Liver weights were increased in males and at the observed magnitude of the effect (relative increase of 27% at 750 mg/kg/day) and in absence of correlating microscopic findings, this was considered non adverse.

A test item-related thickened forestomach was observed in males treated at 750 mg/kg/day. The microscopic correlate for this finding was hyperplasia in the forestomach. Hyperplasia in the forestomach was observed in both males and females at 750 mg/kg/day. Based on the low severity (minimal to slight) and the absence of any inflammatory response, the hyperplasia in the forestomach was considered not adverse. This forestomach lesion was considered to be a local response and most likely due to the minor irritating properties of the test item.  

Parental toxicity: NOEL = 250 mg/kg bw/day. The main observed effects involve:

1. consistent effects that suggest local irritation:

- Dose related salivation, starting at 250 mg/kg bw

- Consistent slightly lower food intake and BW-gain at 750 mg/kg.

- forestomach effects in 4/5 males and 3/5 females at 750 mg/kg.

2. increase liver weight at least in males, suggesting liver induction involving change of epoxide into diols that are readily further metabolized, as first pass metabolism after uptake. These effects are consistent with adverse effects observed after only 14 days treatment in RF.

These effects were however not considered adverse, resulting to a NOAEL of 750 mg/kg bw/day.

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Jan 2019 - 15 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Purity/Composition correction factor: No correction factor required (substance is a UVCB)
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks old
- Weight at study initiation: between 209 and 248 g (females)
- Fasting period before study: no
- Housing: On arrival and following 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 Macrolon plastic cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages), with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage. The cages contained appropriate bedding (Lignocel S8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. During motor activity measurements, animals had no access to food or water for a maximum of 2 hours.
- Water: Municipal tap water, ad libitum.
- Acclimation period: 8 days
DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 45 to 49%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES:
From 31 Jan 2019 to 8 Apr 2019
Route of administration:
oral: gavage
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Dose volume: 5 mL/kg.
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 to 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 67 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40 to 43 days.
Two females from the control group and 2 females from the mid dose group were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The dosing formulations were stirred continuously during dose administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (approximately 500 mg) were analysed for concentration and homogeneity. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of target concentration. Homogeneity 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 and demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
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 postcoitum. Once mating had occurred, the males and females were separated. Daily vaginal lavage was performed for all females during mating until evidence of copulation was observed. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. Each non-mated female was re-mated once with a male of proven fertility of the same group for 1 day.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 to 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 67 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40 to 43 days. Two females from the control group and 2 females from the mid dose group were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily
Duration of test:
Terminated at end of exposure
Dose / conc.:
83 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected based on the results of a dose range finder study. Three females were dosed once daily by oral gavage at 500 mg/kg bw/day, and 3 females were dosed at 1000 mg/kg bw/day for 7 days a week for 14 days. The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg bw/day). Mortality was checked twice daily throughout the study. Clinical Observations were done at least daily up to the day prior to necropsy, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights were measured on Day 1 prior to dosing and on Days 4, 8, 11 and 14. Food Consumption was monito
red over Days 1-4, 4-8, 8-11 and 11-14. All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body
weight, kidney and liver weight were determined at scheduled necropsy.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Twice daily, in the morning and at the end of the working day.
DETAILED CLINICAL OBSERVATIONS: Yes
- Once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy. During the dosing period, these observations were perform
ed after dosing at no specific time point, but within a similar time period after dosing for the respective animals. The peak effect of occurrence of clinical signs occurred directly after dosing. Therefore,
clinical observations and functional observation tests were conducted directly after dosing.
BODY WEIGHT: Yes
- 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: Yes
- Food consumption was quantitatively measured weekly, except for females which were housed together for mating and for females without evidence of mating. 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.
WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling,
water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean
corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells Platelets, Reticulocytes (absolute).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate.
Assessment of T4 and Thyroid Stimulating Hormone (TSH) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded. In addition, coagulation parameters were analysed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).
NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after completion of clinical observations. The following tests were performed: Hearing ability, pupillary reflex, static righting reflex.
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal;
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Ovaries and uterine content:
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Weight of uterus (weighed with cervix) and ovaries were determined at necropsy for all selected animals.
Fetal examinations:
Culling performed on PND 4. Eight pups from each litter of equal sex distribution (if possible) will be selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, will not be done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable.

The number of live and dead pups will be determined on PND 1 and daily thereafter. Pups will be observed for general health/mortality and moribundity. Pups will not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Detailed clinical observations will be made for all pups at least once daily. Live pups will be individually weighed on PND 1, 4, 7, and 13. Sex will be externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) will be measured for all live pups on PND 1. The AGD will be normalized to the cube root of body weight.

All males in each litter will be examined for the number of areola/nipples on PND 13.
GROSS EXAMINATION OF DEAD PUPS:
Pups that die or are euthanized before scheduled termination will be examined externally and sexed (both externally and internally, if possible). All remaining pups will be euthanized on PND 14-16. Sex will be determined both externally and internally. Descriptions of all external abnormalities will be recorded. Particular attention will be paid to the external reproductive genitals to examine signs of altered development. External abnormalities may be collected and fixed in 10% buffered formalin. In addition, the thyroid will be collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection), and will be preserved in 10% buffered formalin.
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 to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made: Low dose group vs. Control group; mid dose group vs. Control group; High dose group vs. Control group.
Parametric: 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 was 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.
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
- 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 pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Historical control data:
Historical data present at test facility for tests performed between 2015-2018. Where relevant, historical reference data are included in the report (with the number of tests used).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations up to 83 mg/kg bw/day (low dose group). No findings were noted during the weekly arena observations in this study. Salivation was seen after dosing among animals of the 250 (mid dose group) and 750 mg/kg bw/day (high dose group) from the first week of the treatment period onwards and was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
One female of the 750 mg/kg bw/day (high dose group) was euthanized on Lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in body weights and body weight gain were observed up to 250 mg/kg bw/day (mid dose group). At Day 8 of pre mating period, body weight gain in males at 750 mg/kg bw/day (high dose group) was decreased to 0.78x when compared with control. During the following weeks of treatment, body weight gains were considered normal. At the end of the post coitum and lactation period, lower (though non-statistically significant) body weights and body weight gains were observed in females at 750 mg/kg bw/day. In absence of statistically significant findings, these changes in females were considered non adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level up to 250 mg/kg bw/day (mid dose group). In males at 750 mg/kg bw/day (high dose group), absolute and relative food consumption was slightly lower compared with controls (0.92x and 0.96x, respectively) during the first week of treatment. In the following weeks, food consumption levels were considered normal.
In females at 750 mg/kg bw/day, absolute food consumption was decreased, when compared with control during the first week of treatment and during the lactation period (0.88x and 0.74-0.76x, respectively), but not during the post coitum period. Similar decreases in relative food consumption were observed during the first 2 weeks of treatment (0.84-0.91x) and the lactation period (0.74-0.77x). Also, relative food consumption levels were decreased during the first three weeks of the post coitum period (0.86-0.91x). 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.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment up to 750 mg/kg bw/day (high dose group). For males, the available data of low and high dose groups indicate that there is no dose related trend and that results are within normal range when compared with the historical control data (For males only one control measurement was available, therefore low and high dose groups data was compared with historical control data). In females, any statistically significant changes noted in hematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters of treated rats were considered unaffected by treatment up to 83 mg/kg bw/day (low dose group). In males, albumin levels were increased with 1.04x and 1.06x at 250 and 750 mg/kg bw/day (mid and high dose groups), respectively, when compared to control, and urea levels were increased with 1.29x at 750 mg/kg bw/day. In females, bile acids levels were increased with 2.88x at 750 mg/kg bw/day. As all values remained within normal range, these changes were considered not toxicologically relevant. Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response.
Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment.
Coagulation parameters: Unaffected by treatment up to 750 mg/kg bw/day.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment up to 750 mg/kg bw/day (high dose group). Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval and a decreasing trend in activity over the duration of the test period. All values remained within normal range.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in one high dose female, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight hyperplasia of the forestomach was recorded in 2 females of the 750 mg/kg bw/day group (high dose group). 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.
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The number of implantation sites was statistically significantly decreased for the high dose group (mean implantation sites: 14.2, 13.3, 12.0 and 9.4 for control, low, mid and high dose group, respectively).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter loss was observed by resorption.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The average duration of gestation was 21.6, 21.8, 21.6 and 21.4 days for the control groups and the groups dosed at 83, 250 and 750 mg/kg bw/day, respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The gestation indices for all groups were 100%.
Details on maternal toxic effects:
Mating index was considered not to be affected by treatment (100% for all groups). Precoital time was considered unaffected by treatment. With the exception of two females at dose 83 mg/kg bw/day, all females showed evidence of mating within 14 days.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects seen up to and including highest dose tested (750 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Reproductive performance
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were considered unaffected by treatment with the test item up to 250 mg/kg bw/day. From PND 1, a trend towards lower body weights in male and female pups was observed at 750 mg/kg bw/day, reaching statistical significance at PND 13 (decreased to 0.83x and 0.86x, respectively, when compared with control).
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
One pup of the control group, one pup at 83 mg/kg bw/day and 3 pups at 750 mg/kg bw/day were missing on PND 2 or 3. Pups missing were most likely cannibalised. One pup at 83 mg/kg bw/day, was sacrificed for humane reasons on PND 1. No toxicological relevance was attributed to these sacrificed/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 in the high dose group had total litter loss on PND 2.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered unaffected by treatment with the test item up to 750 mg/kg bw/day. At 250 mg/kg bw/day, more females than males were present. In absence of a dose related trend, this shift in male/female ratio was considered unrelated to treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The viability index was 99%, 98%, 100% and 96% for the control, low, mid and high dose, respectively.
The lactation index was 100%, 98%, 100% and 98% for the control, low, mid and high dose, respectively.

External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item up to 750 mg/kg bw/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.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered unaffected by treatment up to 750 mg/kg bw/day. The statistically significantly higher mean anogenital distance of female pups at 83 mg/kg bw/day occurred without a dose related-trend. This variation was considered not to be related to treatment. Treatment with the test item up to 750 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 14-16 pups were considered unaffected by treatment with the test item up to 750 mg/kg bw/day. No statistically significant changes were noted in Total T4 levels of male and female PND 14-16 pups and all values were considered within normal range.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Results Dose Formulation Analyses:

The concentrations analyzed in the formulations of the low , mid and high dose groups were in agreement with target concentrations (i.e. mean accuracies between 96% and 99%). No test item was detected in

the Control group formulation. The formulations of the low dose group and the high dose group were homogeneous (i.e. coefficient of variation ≤ 2.0%).

Results dose range finder study:

No mortality occurred. At 1000 mg/kg bw/day, piloerection was present in all animals during Days 2-8. Hunched posture was present in 2-3/3 animals from Days 3-8 and from Days 11-14. Salivation was

observed in 3/3 animals on most days during the second week of treatment. On Day 7, lethargy was observed in 2/3 animals. Body weight was reduced in 1/3 animals during Days 1-4, followed by normal body weights and body weight gain. During Days 1-4, absolute and relative food consumption was reduced. At 500 mg/kg bw/day, a hunched posture was observed in 1/3 animals on Days 13 and 14. Piloerection was seen in 3/3 animals

on Day 8. Salivation was observed in 3/3 animals on most days during the second week of treatment. Normal body weights and body weight gain was measured, food consumption was considered within

normal range. At necropsy, no abnormalities were noted at 500 mg/kg bw/day. Kidney weights were within normal range, but relative liver weights were increased with approximately 16% compared with the historical

control range. At 1000 mg/kg bw/day, in 1/3 animals the stomach was grown together with the liver and the liver was grown together with the diaphragm. No effects were seen on kidney weights, relative liver

weights were increased with approximately 39% compared with the historical control range.

Conclusions:
A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. C12-14 Alkyl ethoxylated glycidylether was administered by daily oral gavage to male and female rats at dose levels of 83, 250 and 750 mg/kg bw/day. Based on the results of this study, the parental NOEL was concluded 250 mg/kg/day (based on lower body weight gain and food consumption, increased liver weights in males, a thickened forestomach in males and hyperplasia in the forestomach in males and females at 750 mg/kg/day). As these effects were considered to be non-adverse, the NOAEL was found to be 750 mg/kg bw/day. Based on decreased postnatal body weight in pups at 750 mg/kg bw/day, the Developmental NOAEL was found to be 250 mg/kg bw/day.
Executive summary:

C12-14 Alkyl ethoxylated glycidylether has been evaluated in a combined 28-day repeated dose toxicity with reproduction/developmental toxicity screening in rats. The animals were dosed for 29-32 days (males) and average 54 days (females, range 50-67 days and 40-43 days in case non-pregnant or total litter loss).

The applied dose levels of 0, 83, 250, 750 mg/kg/day were based on results of a 14-day range finder involving two groups of 3 female rats at 500 and 1000 mg/kg/day. Effects at 1000 mg involved: hunched posture and lethargy; BW reduction in one animal; reduction absolute and relative food consumption; effects on stomach in one animal at macroscopic examination; increase relative liver weights by 39%.

The following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.  In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).  

Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

The observed decrease in body weight gain in males at 750 mg/kg/day on Day 8, was considered non adverse as body weights were normal and the body weight gain recovered to normal levels in the following weeks. The decrease in body weight gain may have been related to the decreased food consumption levels found during the first week of treatment in males at 750 mg/kg/day.

For females, (relative) food consumption levels were decreased during the premating, post coitum and/or lactation period at 750 mg/kg/day. Though not statistically significant, body weight gains were lower at the end of post coitum and lactation period at 750 mg/kg/day. These findings were likely the result of the decreased litter sizes at 750 mg/kg/day and were considered secondary to developmental and reproductive toxicity.

Liver weights were increased in males and at the observed magnitude of the effect (relative increase of 27% at 750 mg/kg/day) and in absence of correlating microscopic findings, this was considered non adverse.

A test item-related thickened forestomach was observed in males treated at 750 mg/kg/day. The microscopic correlate for this finding was hyperplasia in the forestomach. Hyperplasia in the forestomach was observed in both males and females at 750 mg/kg/day. Based on the low severity (minimal to slight) and the absence of any inflammatory response, the hyperplasia in the forestomach was considered not adverse. This forestomach lesion was considered to be a local response and most likely due to the minor irritating properties of the test item.  

Parental toxicity: NOEL = 250 mg/kg bw/day. The main observed effects involve:

1. consistent effects that suggest local irritation:

- Dose related salivation, starting at 250 mg/kg bw

- Consistent slightly lower food intake and BW-gain at 750 mg/kg.

- forestomach effects in 4/5 males and 3/5 females at 750 mg/kg.

2. increase liver weight at least in males, suggesting liver induction involving change of epoxide into diols that are readily further metabolized, as first pass metabolism after uptake. These effects are consistent with adverse effects observed after only 14 days treatment in RF.

These effects were however not considered adverse, resulting to a NOAEL of 750 mg/kg bw/day.

Developmental effects: The only effect observed is a slightly lower BW at birth, and a lower BW gain of pups thereafter in the 750 mg group only. This is fully consistent with lower food intake/growth seen in the parents, possibly amplified as in lactation phase the food intake of the mothers can be more than double the normal amount. As such it is likely a secondary effect, and not a direct acting developmental toxicant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test.
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test.
Version / remarks:
2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents.
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: clear colourless liquid
- Storage conditions: at room temperature
- Test item name as cited in the report: C12-14 Alcohol ethoxylated (EO<2.5), reaction products with epichlorohydrin
- Purity: UVCB
- Purity test date: 21 Sept 2018
- Batch: 1658866
- Expiry date of batch: 15 August 2019
Specific details on test material used for the study:
Purity/Composition correction factor: No correction factor required (substance is a UVCB)

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks old (males), 13-14 weeks old (females)
- Weight at study initiation: between 261 and 313 g (males), between 209 and 248 g (females)
- Fasting period before study: no
- Housing: On arrival and following 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 Macrolon plastic cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages), with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. During motor activity measurements, animals had no access to food or water for a maximum of 2 hours.
- Water: Municipal tap water, ad libitum.
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 45 to 49%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From 31 Jan 2019 to 8 Apr 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Dose volume: 5 mL/kg.
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 to 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 67 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40 to 43 days.
2 females from the control group and 2 females from the mid dose group were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
The dose volume for each animal was based on the most recent body weight measurement.
The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
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. Daily vaginal lavage was performed for all females during mating until evidence of copulation was observed. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. Each non-mated female was re-mated once with a male of proven fertility of the same group for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (approximately 500 mg) were analysed for concentration and homogeneity. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of target concentration. Homogeneity 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 and demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 to 32 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50 to 67 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40 to 43 days. Two females from the control group and 2 females from the mid dose group were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
83 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected based on the results of a dose range finder study. Three females were dosed once daily by oral gavage at 500 mg/kg bw/day, and 3 females were dosed at 1000 mg/kg bw/day for 7 days a week for 14 days. The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg bw/day). Mortality was checked twice daily throughout the study. Clinical Observations were done at least daily up to the day prior to necropsy, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights were measured on Day 1 prior to dosing and on Days 4, 8, 11 and 14. Food Consumption was monitored over Days 1-4, 4-8, 8-11 and 11-14. All animals were subjected to an external, thoracic and abdominal examination on Day 15 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body
weight, kidney and liver weight were determined at scheduled necropsy.
Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once daily, beginning during the first administration of the test item and lasting throughout the dosing period up to the day prior to necropsy. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals. The peak effect of occurrence of clinical signs occurred directly after dosing. Therefore, clinical observations and functional observation tests were conducted directly after dosing.

BODY WEIGHT: Yes
- Animals were weighed individually on the first day of treatment (prior to dosing), and weekly the reafter. 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: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. 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.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells Platelets, Reticulocytes (absolute).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Males were fasted overnight with a maximum of 24 hours before blood sampling, water was available; females were not fasted.
- How many animals: all animals, except for one female with total litter loss.
- Parameters checked: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Pota
ssium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate.

Serum was analyzed for total Thyroxine (T4) for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

In addition, coagulation parameters were analysed: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after completion of clinical observations. The following tests were performed: Hearing ability, pupillary reflex, static righting reflex.
- Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal;
- Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
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):
For the testes of all selected males of the groups, and all males that fail to sire or died before mating detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination will be conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings will be noted.
Litter observations:
The females will be allowed to litter normally. Cage debris of pregnant females will be examined for evidence of premature delivery. Signs of difficult or prolonged parturition will be recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, will be recorded, if applicable.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Eight pups from each litter of equal sex distribution (if possible) will be selected. Blood samples will be collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, will not be done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) is acceptable.

PARAMETERS EXAMINED
The number of live and dead pups will be determined on PND 1 and daily thereafter. Pups will be observed for general health/mortality and moribundity. Pups will not be removed from the cage during observation, unless necessary for identification or confirmation of possible findings.
Detailed clinical observations will be made for all pups at least once daily. Live pups will be individually weighed on PND 1, 4, 7, and 13. Sex will be externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) will be measured for all live pups on PND 1. The AGD will be normalized to the cube root of body weight.
All males in each litter will be examined for the number of areola/nipples on PND 13.

GROSS EXAMINATION OF DEAD PUPS:
Pups that die or are euthanized before scheduled termination will be examined externally and sexed (both externally and internally, if possible).
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
ORGAN WEIGHTS
- On selected 5 animals/sex/group: according to guidelines.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.
HISTOPATHOLOGY: Yes
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: according to guidelines.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups:
Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle,and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.
- For the testes of all selected males of the low and high dose groups, and one male of the control group, a detailed qualitative examination was made, taking into account the tubular stages of the
spermatogenic cycle.
Postmortem examinations (offspring):
All remaining pups will be euthanized on PND 14-16. Sex will be determined both externally and internally. Descriptions of all external abnormalities will be recorded. Particular attention will be paid to the external reproductive genitals to examine signs of altered development. External abnormalities may be collected and fixed in 10% buffered formalin. In addition, the thyroid will be collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for (complete) blood collection), and will be preserved in 10% buffered formalin.
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 to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made: Low dose group vs. Control group; mid dose group vs. Control group; High dose group vs. Control group.
Parametric: 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 was applied with Bonferroni correction for multiple test ing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above 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:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- 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 pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were noted during daily detailed clinical observations up to 83 mg/ kg bw/day (low dose group). No findings were noted during the weekly arena observations in this study. Salivation was seen after dosing among animals of the 250 (mid dose group) and 750 mg/kg bw/day (high dose group) from the first week of the treatment period onwards and was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
One female of the 750 mg/kg bw/day (high dose group) was euthanized on Lactation Day 2, as she had a total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related changes in body weights and body weight gain were observed up to 250 mg/kg bw/day (mid dose group). At Day 8 of pre mating period, body weight gain in males at 750 mg/kg bw/day (high dose group) was decreased to 0.78x when compared with control. During the following weeks of treatment, body weight gains were considered normal. At the end of the post coitum and lactation period, lower (though non-statistically significant) body weights and body weight gains were observed in females at 750 mg/kg bw/day. In absence of statistically significant findings, these changes were considered non adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was similar to the control level up to 250 mg/kg bw/day (mid dose group). In males at 750 mg/kg bw/day (high dose group), absolute and relative food consumption was slightly lower compared with controls (0.92x and 0.96x, respectively) during the first week of treatment. In the following weeks, food consumption levels were considered normal. In females at 750 mg/kg bw/day, absolute food consumption was decreased, when compared with control during the first week of treatment and during the lactation period (0.88x and 0.74-0.76x, respectively), but not during the post coitum period. Similar decreases in relative food consumption were observed during the first 2 weeks of treatment (0.84-0.91x) and the lactation period (0.74-0.77x). Also, relative food consumption levels were decreased during the first three weeks of the post coitum period (0.86-0.91x). 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.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment up to 750 mg/ kg bw/day (high dose group). For males, the available data of low and high dose groups indicate that there is no dose related trend and that results are within normal range when compared with the historical control data (For males only one control measurement was available, therefore low and high dose groups data was compared with historical control data). In females, any statistically significant changes noted in hematology parameters achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose response.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters of treated rats were considered unaffected by treatment up to 83 mg/kg bw/day (low dose group). In males, albumin levels were increased with 1.04x and 1.06x at 250 and
750 mg/kg bw/day (mid and high dose groups), respectively, when compared to control, and urea levels were increased with 1.29x at 750 mg/kg bw/day. In females, bile acids levels were increased with 2.88x
at 750 mg/kg bw/day. As all values remained within normal range, these changes were considered not toxicologically relevant. Any other statistically significant changes in clinical chemistry parameters
achieving a level of statistical significance when compared to controls were considered unrelated to administration of the test item due to the minimal magnitude of the change and/or absence of a dose
response.
Thyroid hormone analyses: Serum levels of T4 in F0-males were considered unaffected by treatment. Coagulation parameters: unaffected by treatment up to 750 mg/kg bw/day. For males, the available da
ta of 250 and 750 mg/kg bw/day (mid and high dose groups) indicate that there is no dose related trend and that results are within normal range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered unaffected by treatment up to 750 mg/kg bw/day (high dose group). Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval and a decreasing trend in activity over the duration of the test period. All values remained within normal range.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher liver weights (absolute and relative to body weights) were noted in the 750 mg/kg bw/day group males (high dose group). There was no microscopic correlate with the increased liver weight. Some organ weight differences were statistically significant when compared to the control group (heart, liver, kidneys in males; relative to body weight only) but were considered to be the result of a test item-related effect on final body weight. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related thickened forestomach was observed in the stomach of 2/10 males treated at 750 mg/kg bw/day (high dose group). The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in one high dose female, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight hyperplasia of the forestomach was recorded in 3 males and 2 females of the 750 mg/kg bw/day group (high dose group). 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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered unaffected by treatment. Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 750 mg/kg bw/day (with normal litter). Given their incidental nature, absence of a doserelated incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis of two males of the low dose group that failed to sire 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, treatment-related
Description (incidence and severity):
The number of implantation sites was statistically significantly decreased for the high dose group (mean implantation sites: 14.2, 13.3, 12.0 and 9.4 for control, low, mid and high dose group, respectively).

Mating index was considered not to be affected by treatment (100% for all groups). Precoital time was considered unaffected by treatment. With the exception of two females at dose 83 mg/kg bw/day (males failed to sire), all females showed evidence of mating within 14 days. The fertility indices for the control, low, mid and high dose group were 100%, 80%, 90% and 90%, respectively. One female in the mid dose group was not pregnant, and two females were not pregnant in the high dose group. There were no morphological findings in the reproductive organs of either sex which could explain the failure of sire or delivering healthy pups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including highest dose tested (750 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
other: Male and/or female reproduction system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

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. One pup in a nest of the low dose group was found cold, with cramps and a purple color and absence of milk in the stomach and several wounds in the abdomen were found on Day 1. The pups of one female from the high dose group which were missing on Day 2, were found cold, and absence of milk in the stomach was noted on Day 1. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One pup of the control group, one pup at 83 mg/kg bw/day and 3 pups at 750 mg/kg bw/day were missing on PND 2 or 3. Pups missing were most likely cannibalised. One pup at 83 mg/kg bw/day, was sacrificed for humane reasons on PND 1. No toxicological relevance was attributed to these sacrificed/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 in the high dose group had total litter loss on PND 2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of pups were considered unaffected by treatment with the test item up to 250 mg/kg bw/day. From PND 1, a trend towards lower body weights in male and female pups was observed at 750 mg/kg bw/day, reaching statistical significance at PND 13 (decreased to 0.83x and 0.86x, respectively, when compared with control).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered unaffected by treatment with the test item up to 750 mg/kg bw/day. No statistically significant changes were noted in Total T4 levels of male and female PND 14-16 pups and all values were considered within normal range (Historical control data for PND 14-16 Wistar Han rats (period 2017-2019): Total T4 (μg/dL) (males): mean = 5.93; P5 - P95 = 4.28 – 8.03 (n=499), Total T4 (μg/dL) (females): mean = 5.57; P5 - P95 = 4.04 – 7.50 (n=499)).
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered unaffected by treatment up to 750 mg/kg bw/day. The statistically significantly higher mean anogenital distance of female pups at 83 mg/kg bw/day occurred without a dose related-trend. This variation was considered not to be related to treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 750 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item up to 750 mg/kg bw/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.
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio was considered unaffected by treatment with the test item up to 750 mg/kg bw/day. At 250 mg/kg bw/day, more females than males were present. In absence of a dose related trend, this shift in male/female ratio was considered unrelated to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
other: general toxicity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw (total dose)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Results Dose Formulation Analyses:

The concentrations analyzed in the formulations of the low , mid and high dose groups were in agreement with target concentrations (i.e. mean accuracies between 96% and 99%). No test item was detected in the Control group formulation. The formulations of the low dose group and the high dose group were homogeneous (i.e. coefficient of variation ≤ 2.0%).

Results dose range finder study:

No mortality occurred. At 1000 mg/kg bw/day, piloerection was present in all animals during Days 2-8. Hunched posture was present in 2-3/3 animals from Days 3-8 and from Days 11-14. Salivation was observed in 3/3 animals on most days during the second week of treatment. On Day 7, lethargy was observed in 2/3 animals. Body weight was reduced in 1/3 animals during Days 1-4, followed by normal body weights and body weight gain. During Days 1-4, absolute and relative food consumption was reduced. At 500 mg/kg bw/day, a hunched posture was observed in 1/3 animals on Days 13 and 14. Piloerection was seen in 3/3 animals on Day 8. Salivation was observed in 3/3 animals on most days during the second week of treatment. Normal body weights and body weight gain was measured, food consumption was considered within normal range. At necropsy, no abnormalities were noted at 500 mg/kg bw/day. Kidney weights were within normal range, but relative liver weights were increased with approximately 16% compared with the historical control range. At 1000 mg/kg bw/day, in 1/3 animals the stomach was grown together with the liver and the liver was grown together with the diaphragm. No effects were seen on kidney weights, relative liver weights were increased with approximately 39% compared with the historical control range.

Applicant's summary and conclusion

Conclusions:
A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. C12-14 Alkyl ethoxylated glycidylether was administered by daily oral gavage to male and female rats at dose levels of 83, 250 and 750 mg/kg bw/day. Based on the results of this study, the parental NOEL was concluded 250 mg/kg/day (based on lower body weight gain and food consumption, increased liver weights in males, a thickened forestomach in males and hyperplasia in the forestomach in males and females at 750 mg/kg/day). As these effects were considered to be non-adverse, the NOAEL was found to be 750 mg/kg bw/day. Based on a decreased number of implantation sites at 750 mg/kg bw/day, the Reproduction NOAEL was found to be 250 mg/kg bw/day.
Executive summary:

C12-14 Alkyl ethoxylated glycidylether has been evaluated in a combined 28-day repeated dose toxicity with reproduction/developmental toxicity screening in rats. The animals were dosed for 29-32 days (males) and average 54 days (females, range 50-67 days and 40-43 days in case non-pregnant or total litter loss).

The applied dose levels of 0, 83, 250, 750 mg/kg/day were based on results of a 14-day range finder involving two groups of 3 female rats at 500 and 1000 mg/kg/day. Effects at 1000 mg involved: hunched posture and lethargy; BW reduction in one animal; reduction absolute and relative food consumption; effects on stomach in one animal at macroscopic examination; increase relative liver weights by 39%.

The following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.  In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).  

Formulation analyses confirmed that formulations of test item in water were prepared accurately and homogenously.

The observed decrease in body weight gain in males at 750 mg/kg/day on Day 8, was considered non adverse as body weights were normal and the body weight gain recovered to normal levels in the following weeks. The decrease in body weight gain may have been related to the decreased food consumption levels found during the first week of treatment in males at 750 mg/kg/day.

For females, (relative) food consumption levels were decreased during the premating, post coitum and/or lactation period at 750 mg/kg/day. Though not statistically significant, body weight gains were lower at the end of post coitum and lactation period at 750 mg/kg/day. These findings were likely the result of the decreased litter sizes at 750 mg/kg/day and were considered secondary to developmental and reproductive toxicity.

Liver weights were increased in males and at the observed magnitude of the effect (relative increase of 27% at 750 mg/kg/day) and in absence of correlating microscopic findings, this was considered non adverse.

A test item-related thickened forestomach was observed in males treated at 750 mg/kg/day. The microscopic correlate for this finding was hyperplasia in the forestomach. Hyperplasia in the forestomach was observed in both males and females at 750 mg/kg/day. Based on the low severity (minimal to slight) and the absence of any inflammatory response, the hyperplasia in the forestomach was considered not adverse. This forestomach lesion was considered to be a local response and most likely due to the minor irritating properties of the test item.  

Parental toxicity: NOEL = 250 mg/kg bw/day. The main observed effects involve:

1. consistent effects that suggest local irritation:

- Dose related salivation, starting at 250 mg/kg bw

- Consistent slightly lower food intake and BW-gain at 750 mg/kg.

- forestomach effects in 4/5 males and 3/5 females at 750 mg/kg.

2. increase liver weight at least in males, suggesting liver induction involving change of epoxide into diols that are readily further metabolized, as first pass metabolism after uptake. These effects are consistent with adverse effects observed after only 14 days treatment in RF.

These effects were however not considered adverse, resulting to a NOAEL of 750 mg/kg bw/day.

Reproductive effects: A lower number of implantation sites was observed at 750 mg that was statistically significant different from control, but not significantly different compared historical. No corpora lutea were counted, so whether this is based on lower number of ovulations or resulting from a relative increase of pre-implantation loss cannot be distinguished. Otherwise there were no effects on reproduction.