Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sept 2018 - 21 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sept 2018 - 21 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks (males), 13-14 weeks (females).
- Weight at study initiation: 257-305 g (males) or 203 - 231 g (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. During the lactation phase, females were housed in Macrolon plastic cages.
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).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 46 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Sept 2018 to 21 Nov 2018

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of formulation: 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 weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For concentration analysis, results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Duplicate middle samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For homogeneity anaylsis, results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%. Stability analyses performed previously in conjunction with the method development and validation study 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:

Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of high dose group), 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.
The first day of dosing was designated as Day 1. One female from the control group, one in the mid dose group and one in the high 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, 7 d/w

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

1 000 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 dose levels were selected based on the results of a 10-day dose range finder with oral administration of Reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol in rats, and to determine the peak effect of occurrence of clinical signs after dosing. This dose range finding study was performed with staggered start, as no in vivo data was available for the reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol. The first dose level was 500 mg/kg bw/day (low dose group), based on an acute toxicity end point summary that supports an oral LD50 of >2000mg/kg with QSAR data and the absence of cytotoxicity in the Neural Red 3t3 cell test. The second dose level was 1000 mg/kg bw/day (high dose group), it was selected based on the lack of toxicologically relevant changes in the low dose group up to Day 6 of treatment. Mortality was observed twice daily during the study. Clinical observations were performed at least daily from days 1-10 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 5 and 10. Food consumption was recorded for the high dose group over days 1-5, and for the low dose group over days 3-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11. Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

Positive control:

No.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed 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 postnatal Days 1, 4, 7 and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females)

FOOD CONSUMPTION:
Yes. Weekly, for males and females, 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
- Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION:
No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females not.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females no.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 8-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported.

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 13 days prior to treatment, 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. This was done for all females, except for one female from the control group that had to be euthanized in extremis.

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 comparison 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, mid dose group vs. control, and high dose group vs. control.
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).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
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:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 19 of post-coitum, one female from the control group was sacrificed as she expressed severe clinical symptoms including lethargy, a flat and hunched posture, slow breathing, piloerection, hypothermia, partially closed eyes (i.e. ptosis), and a dehydrated and pale appearance. At macroscopic examination, main findings were a pale appearance of the animal, an enlarged liver, gelatinous pancreas, enlarged iliac and renal lymph nodes, and a red discolouration of the mesenteric and renal lymph nodes The uterus contained in total 12 dead fetuses. Microscopic examination revealed marked necrosis of the liver (correlating with a macroscopically enlarged liver) and marked tubular degeneration of the kidneys. These alterations in liver and kidney were regarded the main cause of moribundity for this animal.
On the day of scheduled necropsy, one female from the high dose group died after the blood sampling procedure. There were no microscopic findings of note for this animal and this death was regarded to be related to the blood sampling procedure under anaesthesia and therefore unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight of treated females at 1000 mg/kg bw/day was statistically significantly increased (4.7%) when compared with concurrent control on Day 1 of dosing and remained slightly higher throughout treatment. As the body weight gain of these females remained similar to control values, the higher mean body weight on Day 1 was attributed to biological variation (females were allocated prior to start pretest and thus at least two weeks prior to the first administration of test item).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decrease in mean corpuscular volume (MCV) of 4% (mean 52.1 ± 2.0, n=5; control mean 54.5± 1.3, n=5 ) and in mean corpuscular haemoglobin (MCH) of 6% (mean 1.7 ± 0.04, n=5; control mean 1.14 ± 0.05, n=5) compared with the concurrent control was noted in males at 300 mg/kg bw/day. These changes were considered unrelated to administration of the test item due to the minimal magnitude of the change and absence of a dose response.
In females at 1000 mg/kg bw/day, the mean eosinophils level was statistically significantly decreased (mean 0.0 ± 0.0, n=4). As values remained within the historical control range (Historical control data for eosinophils in female Wistar Han rats period 2017-2018 (10E9/L): mean: 0.1; P5-P95: 0.00-0.13, n=206), this finding was considered unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistical signifance noted for mean albumin levels of males at 100 mg/kg bw/day (mean 32.0 ± 0.7, n=5) and 1000 mg/kg bw/day (mean 32.7 ± 0.6, n=5) were attributed to a relatively high concurrent control value (control mean 33.8 ± 0.7, n=5) and therefore considered unrelated to treatment with the test item. (Historical control data for albumin (g/L) in male Wistar han rats for period 2017-2018: mean: 32.2, P5 – P95: 30.2 – 34.0, n=270).
Other statistically significant changes in clinical biochemistry parameters of males at 100 mg/kg bw/day, i.e. total protein (mean 63.0 ± 1.6, n=5; control mean 66.7 ± 2.5 n=5) and potassium (mean 3.62 ± 0.24, n=5; control mean 4.00 ± 0.20, n=5) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses: Serum levels of T4 in F0-males were not considered to be affected by treatment.
Coagulation parameters of treated rats were considered not to have been affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In males, mean grip strength of the fore legs was decreased with 11% at 1000 mg/kg bw/day (mean 1279 ± 100, n=5) when compared with concurrent control (mean 1439 ± 157, n=5). As no statistical significance was achieved, all values remained well within the historical control data range and the concurrent control mean was relatively high, this change was attributed to biological variation (Historical control data for forlimb grip strength (gram) in male Wistar Han rats period 2015-2018: mean = 1158, P5 - P95 = 681 - 1606, n=445). Grip strength values in females were unremarkable. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean relative spleen weight of females at 1000 mg/kg bw/day was deceased with 12% (mean 0.167 ± 0.007, n=4) when compared with concurrent control (mean 0.190 ± 0.028, n=5) . As no statistical significance was achieved and as all values remained well within the historical control range, this change was considered not to be toxicologically relevant. (Historical control data for relative spleen weight (%) in female Wistar han rats, period 2017-2018: mean: 0.174, P5 – P95: 0.145 – 0.210, n=205).
The statistical significance noted for the mean absolute thymus weight of females at 100 mg/kg bw/day was considered unrelated to treatment in the absence of a dose-related response.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Key result

Critical effects observed:

no

Analysis of dose preparations: In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 93% and 97%). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤10%).

Conclusions:

In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), no parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw day). Therefore, the no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy) ethylacetamide and Glycerol was established to be ≥ 1000 mg/kg bw/day.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD/EC guidelines and GLP principles. Reaction mass of N-[2-2hydroxyethoxy) ethylacetamide and Glycerol was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, i.e. water. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation, for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of the high dose group). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. No test item related mortality occurred. Treatment up to 1000 mg/kg bw/day was well tolerated as indicated by the absence of treatment related changes in any of the parental parameters examined (i.e. survival, clinical appearance, functional tests, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). Based on the results of this study, the no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy) ethylacetamide and Glycerol was established to be at least 1000 mg/kg bw/day.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sept 2018 - 21 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Deviations:

no

Principles of method if other than guideline:

- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks (males), 13-14 weeks (females).
- Weight at study initiation: 257-305 g (males) or 203 - 231 g (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. During the lactation phase, females were housed in Macrolon plastic cages.
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).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 46 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Sept 2018 to 21 Nov 2018

Route of administration:

oral: gavage

Details on exposure:

Method of formulation: 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 weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For concentration analysis, results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Duplicate middle samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For homogeneity anaylsis, results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%. Stability analyses performed previously in conjunction with the method development and validation study 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 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females that have not shown evidence of mating were separated from their males. Detection of mating was not confirmed at first instance for one female at the high dose group. Evidence of mating was obtained indirectly by delivery of a litter. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of high dose group), 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.
The first day of dosing was designated as Day 1. One female from the control group, one in the mid dose group and one in the high 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. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 29 days
Females: 50-56 days

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

1 000 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 dose levels were selected based on the results of a 10-day dose range finder with oral administration of Reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol in rats, and to determine the peak effect of occurrence of clinical signs after dosing. This dose range finding study was performed with staggered start, as no in vivo data was available for the reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol. The first dose level was 500 mg/kg bw/day (low dose group), based on an acute toxicity end point summary that supports an oral LD50 of >2000mg/kg with QSAR data and the absence of cytotoxicity in the Neural Red 3t3 cell test. The second dose level was 1000 mg/kg bw/day (high dose group), it was selected based on the lack of toxicologically relevant changes in the low dose group up to Day 6 of treatment. Mortality was observed twice daily during the study. Clinical observations were performed at least daily from days 1-10 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 5 and 10. Food consumption was recorded for the high dose group over days 1-5, and for the low dose group over days 3-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11. Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed 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 postnatal Days 1, 4, 7 and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females)

FOOD CONSUMPTION:
Yes. Weekly, for males and females, 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
- Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION:
No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females not.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females no.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 8-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported.

PARTURITION AND MATERNAL CARE
Yes. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on post natal days 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on post natal days 1 and 4.
- Anogenital distance was measured on post natal day 1.
- Areola/nipple retention were examined on post natal day 13.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

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 comparison 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, mid dose group vs. control, and high dose group vs. control.
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).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
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:

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

Developmental indices
- Gestation index: (Number of females with living pups on Day 1/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post implantation survival: (Total number of offsprng 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 offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Historical control data:

Historical control data is indicated in results when necessary.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 19 of post-coitum, one female from the control group was sacrificed as she expressed severe clinical symptoms including lethargy, a flat and hunched posture, slow breathing, piloerection, hypothermia, partially closed eyes (i.e. ptosis), and a dehydrated and pale appearance. At macroscopic examination, main findings were a pale appearance of the animal, an enlarged liver, gelatinous pancreas, enlarged iliac and renal lymph nodes, and a red discolouration of the mesenteric and renal lymph nodes The uterus contained in total 12 dead fetuses. Microscopic examination revealed marked necrosis of the liver (correlating with a macroscopically enlarged liver) and marked tubular degeneration of the kidneys. These alterations in liver and kidney were regarded the main cause of moribundity for this animal. On the day of scheduled necropsy, one female from the high dose group died after the blood sampling procedure. There were no microscopic findings of note for this animal and this death was regarded to be related to the blood sampling procedure under anaesthesia and therefore unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight of treated females at 1000 mg/kg bw/day was statistically significantly increased (4.7%) when compared with concurrent control on Day 1 of dosing and remained slightly higher throughout treatment. As the body weight gain of these females remained similar to control values, the higher mean body weight on Day 1 was attributed to biological variation (females were allocated prior to start pretest and thus at least two weeks prior to the first administration of test item).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, the mean eosinophils level was statistically significantly decreased (mean 0.0 ± 0.0, n=4). As values remained within the historical control range (Historical control data for eosinophils in female Wistar Han rats period 2017-2018 (10E9/L): mean: 0.1; P5-P95: 0.00-0.13, n=206), this finding was considered unrelated to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry and coagulation parameters were considered not to have been affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined females. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean relative spleen weight of females at 1000 mg/kg bw/day was deceased with 12% (mean 0.167 ± 0.007, n=4) when compared with concurrent control (mean 0.190 ± 0.028, n=5) . As no statistical significance was achieved and as all values remained well within the historical control range, this change was considered not to be toxicologically relevant. (Historical control data for relative spleen weight (%) in female Wistar han rats, period 2017-2018: mean: 0.174, P5 – P95: 0.145 – 0.210, n=205).
The statistical significance noted for the mean absolute thymus weight of females at 100 mg/kg bw/day was considered unrelated to treatment in the absence of a dose-related response.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Number of implantation sites was considered not to be affected by treatment.
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 88%, 92%, 85% and 93% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Total litter losses by resorption:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 19 of post-coitum, one female from the control group was sacrificed in extremis. The uterus contained in total 12 dead fetuses.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment. The gestation indices were 100% for all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All mated females were pregnant. The fertility indices were 100% for all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No maternal toxicity was observed up to the highest dose level tested.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100%, 100%, 99% and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup at 300 mg/kg bw/day and 3 pups at 1000 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment. Live litter sizes were 11.9, 12.5, 11.9 and 12.2 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

One pup at 300 mg/kg bw/day and 3 pups at 1000 mg/kg bw/day were found dead at first litter check. One pup of the control group, 3 pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead or missing on PND 2 or 3. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age.
Viability index was considered not to be affected by treatment. Viability indices were 99%, 98%, 100% and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Lactation index was considered not to be affected by treatment. Lactation indices were 99%, 100%, 100% and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), no developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). Therefore, the developmental n no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol was established to be ≥1000 mg/kg bw/day.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD/EC guidelines and GLP principles. Reaction mass of N-[2-2hydroxyethoxy) ethylacetamide and Glycerol was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, i.e. water. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation, for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of the high dose group). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. No test item related mortality occurred. Treatment up to 1000 mg/kg bw/day was well tolerated as indicated by the absence of treatment related changes in any of the parental parameters examined (i.e. survival, clinical appearance, functional tests, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Based on the results of this repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study, the developmental no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy) ethylacetamide and Glycerol was established to be at least 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: other guidance as listed under Principles of method if other than guideline

Principles of method if other than guideline:

- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Reaction mass of N-[2-(2hydroxyethoxy) ethylacetamide and Glycerol
CAS number: 118974-46-2/56-81-5
Clear liquid, pale yellow to yellow
pH: 7.5-9.5 at 10% solution
Specific gravity / density: 1.15
Storage: RT in dark
Production date: 03/02/2018
Expiration date: 03/02/2019

Test animals

Species:

rat

Strain:

other: Crl:WI(Han).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 10-11 weeks (males), 13-14 weeks (females).
- Weight at study initiation: 257-305 g (males) or 203 - 231 g (females)
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. During the lactation phase, females were housed in Macrolon plastic cages.
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).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 46 - 76
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Sept 2018 to 21 Nov 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Method of formulation: 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 weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females that have not shown evidence of mating were separated from their males. Detection of mating was not confirmed at first instance for one female at the high dose group. Evidence of mating was obtained indirectly by delivery of a litter. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For concentration analysis, results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Duplicate middle samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. For homogeneity anaylsis, results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%. Stability analyses performed previously in conjunction with the method development and validation study 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:

Males were treated for 29 days, i.e. 14 days prior to mating, during mating and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of high dose group), 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.
The first day of dosing was designated as Day 1. One female from the control group, one in the mid dose group and one in the high 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. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 d/w.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Mid dose group

Doses / concentrations 3

Dose / conc.:

1 000 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 dose levels were selected based on the results of a 10-day dose range finder with oral administration of Reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol in rats, and to determine the peak effect of occurrence of clinical signs after dosing. This dose range finding study was performed with staggered start, as no in vivo data was available for the reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol. The first dose level was 500 mg/kg bw/day (low dose group), based on an acute toxicity end point summary that supports an oral LD50 of >2000mg/kg with QSAR data and the absence of cytotoxicity in the Neural Red 3t3 cell test. The second dose level was 1000 mg/kg bw/day (high dose group), it was selected based on the lack of toxicologically relevant changes in the low dose group up to Day 6 of treatment. Mortality was observed twice daily during the study. Clinical observations were performed at least daily from days 1-10 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 5 and 10. Food consumption was recorded for the high dose group over days 1-5, and for the low dose group over days 3-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 11. Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy and gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

Positive control:

No.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed 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 postnatal Days 1, 4, 7 and 13. Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females)

FOOD CONSUMPTION:
Yes. Weekly, for males and females, 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.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION:
- Yes, on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION:
No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females not.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes, males overnight (with a maximum of 24 hours). Water was provided. Females no.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: During Week 4 of treatment (males) and during the last week of lactation (females, i.e. PND 8-12). After dosing, after completion of clinical observations.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, and fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported.

PARTURITION AND MATERNAL CARE
Yes. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Estrous cycles was evaluated by examining the vaginal cytology of samples obtained by serial vaginal lavage procedures. Daily vaginal lavage was performed starting at 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus. This was done for all females, except for females that were euthanized in extremis or died spontaneously.

Sperm parameters (parental animals):

For the testes of all selected males of the low and high dose groups, and all males that failed to sire detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on post natal days 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on post natal days 1 and 4.
- Anogenital distance was measured on post natal day 1.
- Areola/nipple retention were examined on post natal day 13.
GROSS EXAMINATION OF DEAD PUPS: Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
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.
- The numbers of former implantation sites were recorded for all paired females.

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 once male of the control group, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

SACRIFICE
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.
All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
Live fetuses from one female euthanized in extremis (from control group) were euthanized by decapitation.

GROSS NECROPSY
Pups that died before scheduled termination were examined externally and sexed (both externally and internally).
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol as they were not necropsied on the same day.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.
Recognizable fetuses of the female that was euthanized in extremis (from control group) were examined externally (fetuses were not sexed).

HISTOPATHOLOGY / ORGAN WEIGTHS: No.

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 comparison 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, mid dose group vs. control, and high dose group vs. control.
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).
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
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
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: (Number of pregnant females/Number of females mated) x 100

Offspring viability indices:

- Gestation index: (Number of females with living pups on Day 1/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post implantation survival: (Total number of offsprng 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 offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

On Day 19 of post-coitum, one female from the control group was sacrificed as she expressed severe clinical symptoms including lethargy, a flat and hunched posture, slow breathing, piloerection, hypothermia, partially closed eyes (i.e. ptosis), and a dehydrated and pale appearance. At macroscopic examination, main findings were a pale appearance of the animal, an enlarged liver, gelatinous pancreas, enlarged iliac and renal lymph nodes, and a red discolouration of the mesenteric and renal lymph nodes The uterus contained in total 12 dead fetuses. Microscopic examination revealed marked necrosis of the liver (correlating with a macroscopically enlarged liver) and marked tubular degeneration of the kidneys. These alterations in liver and kidney were regarded the main cause of moribundity for this animal.
On the day of scheduled necropsy, one female from the high dose group died after the blood sampling procedure. There were no microscopic findings of note for this animal and this death was regarded to be related to the blood sampling procedure under anaesthesia and therefore unrelated to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight of treated females at 1000 mg/kg bw/day was statistically significantly increased (4.7%) when compared with concurrent control on Day 1 of dosing and remained slightly higher throughout treatment. As the body weight gain of these females remained similar to control values, the higher mean body weight on Day 1 was attributed to biological variation (females were allocated prior to start pretest and thus at least two weeks prior to the first administration of test item).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A decrease in mean corpuscular volume (MCV) of 4% (mean 52.1 ± 2.0, n=5; control mean 54.5± 1.3, n=5 ) and in mean corpuscular haemoglobin (MCH) of 6% (mean 1.7 ± 0.04, n=5; control mean 1.14 ± 0.05, n=5) compared with the concurrent control was noted in males at 300 mg/kg bw/day. These changes were considered unrelated to administration of the test item due to the minimal magnitude of the change and absence of a dose response.
In females at 1000 mg/kg bw/day, the mean eosinophils level was statistically significantly decreased (mean 0.0 ± 0.0, n=4). As values remained within the historical control range (Historical control data for eosinophils in female Wistar Han rats period 2017-2018 (10E9/L): mean: 0.1; P5-P95: 0.00-0.13, n=206), this finding was considered unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistical signifance noted for mean albumin levels of males at 100 mg/kg bw/day (mean 32.0 ± 0.7, n=5) and 1000 mg/kg bw/day (mean 32.7 ± 0.6, n=5) were attributed to a relatively high concurrent control value (control mean 33.8 ± 0.7, n=5) and therefore considered unrelated to treatment with the test item. (Historical control data for albumin (g/L) in male Wistar han rats for period 2017-2018: mean: 32.2, P5 – P95: 30.2 – 34.0, n=270).
Other statistically significant changes in clinical biochemistry parameters of males at 100 mg/kg bw/day, i.e. total protein (mean 63.0 ± 1.6, n=5; control mean 66.7 ± 2.5 n=5) and potassium (mean 3.62 ± 0.24, n=5; control mean 4.00 ± 0.20, n=5) were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Thyroid hormone analyses: Serum levels of T4 in F0-males were not considered to be affected by treatment.
Coagulation parameters of treated rats were considered not to have been affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In males, mean grip strength of the fore legs was decreased with 11% at 1000 mg/kg bw/day (mean 1279 ± 100, n=5) when compared with concurrent control (mean 1439 ± 157, n=5). As no statistical significance was achieved, all values remained well within the historical control data range and the concurrent control mean was relatively high, this change was attributed to biological variation (Historical control data for forlimb grip strength (gram) in male Wistar Han rats period 2015-2018: mean = 1158, P5 - P95 = 681 - 1606, n=445). Grip strength values in females were unremarkable. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. Most females had regular cycles of 4 days with the exception of one female in the low dose group for which the regularity could not be determined and one female in the mid dose group, which had an irregular cycle. Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There was one control couple without healthy offspring, because the female was euthanized for ethical reasons on Day 19 post coitum. This animal was found pregnant during macroscopic examination (dead fetuses in the uterus). There were no findings in the reproductive organs of male or the female suggesting infertility.
Mating index was considered not to be affected by treatment. All females showed evidence of mating. The mating indices were 100% for all groups.
Precoital time was considered not to be affected by treatment. Most females mated within the first 4 days of the mating period with the exception of two females, which mated at Day 12 or Day 13.
Number of implantation sites was considered not to be affected by treatment.
Fertility index was considered not to be affected by treatment. All mated females were pregnant. The fertility indices were 100% for all groups.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

For one pup that was missing on PND 3 (from control group), a pale appearance was noted at first litter check. The nature and incidence of this 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):

Litter size was considered not affected by treatment. Live litter sizes were 11.9, 12.5, 11.9 and 12.2 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. One pup at 300 mg/kg bw/day and 3 pups at 1000 mg/kg bw/day were found dead at first litter check. One pup of the control group, 3 pups at 100 mg/kg bw/day and one pup at 1000 mg/kg bw/day were found dead or missing on PND 2 or 3. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age.
Post-implantation survival index was considered not to be affected by treatment. Post implantation survival indices were 88%, 92%, 85% and 93% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. .
Live birth index was considered not to be affected by treatment. The live birth indices were 100%, 100%, 99% and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Viability index was considered not to be affected by treatment. Viability indices were 99%, 98%, 100% and 99% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Lactation index was considered not to be affected by treatment. Lactation indices were 99%, 100%, 100% and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

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 not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment. The gestation indices were 100% for all groups.
Sex ratio was considered not to be affected by treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Analysis of dose preparations:In the control group formulation, no test substance was detected. The concentrations analysed in the formulations of groups exposed to 100, 300 and 1000 mg/kg bw/day were in agreement with target concentrations (i.e. mean accuracies between 93% and 97%). The formulations of the low and high dose group were homogeneous (i.e. coefficient of variation ≤10%).

Applicant's summary and conclusion

Conclusions:

In a repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats (OECD 422), no parental or reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg). Therefore, the parental and reproduction no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy)ethylacetamide and Glycerol was established to be ≥1000 mg/kg bw/day.

Executive summary:

A repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study with rats was performed according to OECD/EC guidelines and GLP principles. Reaction mass of N-[2-2hydroxyethoxy) ethylacetamide and Glycerol was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, i.e. water. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation, for 50-56 days (most females) or 63 days (one female of the mid dose group and one female of the high dose group). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. No test item related mortality occurred. Treatment up to 1000 mg/kg bw/day was well tolerated as indicated by the absence of treatment related changes in any of the parental parameters examined (i.e. survival, clinical appearance, functional tests, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). Based on the results of this repeated dose oral toxicity study combined with a reproduction/developmental toxicity screening study, the parental and reproductive no-observed-adverse-effect level (NOAEL) of the reaction mass of N-[2-(2hydroxyethoxy) ethylacetamide and Glycerol was established to be at least 1000 mg/kg bw/day.