Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (OECD 422), rat: NOAEL fertility > 150 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 May - 26 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
adopted 29 Jul 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:
adopted Jul 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
at room temperature, protected from light
- Solubility and stability of the test substance in water: Stability in water for at least 24 h at room temperature under normal laboratory light conditions and for at least 8 days at 2-8 °C was confirmed over the concentration range 1 to 60 mg/mL.
Species:
rat
Strain:
other:
Remarks:
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 weeks (males) and 13 weeks (females)
- Weight at study initiation: 262 – 301 g (males) and 208 – 255 g (females)
- Housing: Animals were housed in groups of 5/sex in Macrolon type MIV cages (height 18 cm) for pre-mating period. During the mating period, males and females were cohabitated on a 1:1 basis in Macrolon type MIII cages (18 cm height). During the post-mating phase, males were housed in their home cages (Macrolon type MIV, 5 males/cage). Females were housed individually in Macrolon type MIII cages (18 cm height) during the post-mating and lactation phase. The cages contained Lignocel S 8-15 bedding and were enriched with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 49 - 72
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 29 Jun 2018 To: 26 Aug 2018
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Elix
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Dosing formulations were prepared weekly as solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at last 30 minutes before dosing. 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. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle: 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use during treatment. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% were also used for the determination of the homogeneity of the formulations. The samples were analysed via UPLC-MS.
Concentrations analyzed for dose groups 2 (3 mg/mL), 3 (10 mg/mL) and 4 (30 mg/mL) were in agreement with target concentrations (mean accuracies between 90 and 110%). A small response at the retention time of the test item was observed in the chromatograms of the group 1 formulation (0 mg/mL). It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.
Homogeneity analysis revealed that that the formulations of Group 2 (3 mg/mL) and Group 4 (30 mg/mL) were homogenous (coefficient of variation ≤ 10%).
Stability analysis was performed prior to the conduction of the test 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:
Males were treated for 29 days, beginning 14 days prior to mating, throughout the mating period up to and including the day before scheduled necropsy.
Females were treated for 50 – 55 days, except for two females of the 50 mg/kg bw/day group which were treated for 62 – 64 days. The treatment started 14 days pre-mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 – 15 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42 – 52 days.
Frequency of treatment:
daily, 7 days/week
Details on study schedule:
not applicable for an OECD 422 study
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a 10-day dose range-finding study in which female Wistar rats (3 per dose) were treated by oral gavage with test item doses of 150 and 300 mg/kg bw/day once daily for 10 consecutive days.
There was no mortality observed during the study period. Clinical signs of toxicity included hunched posture and piloerection in all treated animals during Days 1-3 and 8-10 in the 150 mg/kg bw/day dose group. Animals administered 300 mg/kg bw/day further displayed salivation between study Days 6-10 and on incidental occasions decreased motor activity and flat gait. Body weight loss of 1% and 5-6% after 10 days of treatment was noted in 2/3 animals administered 150 mg/kg bw/day 300 mg/kg bw/day. Food consumption was reduced throughout the treatment period at 150 mg/kg bw/day and up to Day 5 at 300 mg/kg bw/day, respectively. Food consumption was reduced throughout the treatment period at 150 mg/kg bw/day and up to Day 5 at 300 mg/kg bw/day. Relative food consumption was decreased with 35 and 20% over Days 1-5 and 5-10 in the 150 mg/kg bw/da group and decreased with 25% in the 300 mg/kg bw/day group when compared with historical control data. There were no macroscopic findings, but absolute and relative liver weight were increased in both dose groups. Mean relative liver weight was increased with 6% in the150 mg/kg bw/day group and with 22% in the 300 mg/kg bw/day group when compared with historical control data.
Based on the results of the study, the selected dose levels for the main study were 15, 50 and 150 mg/kg bw/day.

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: general health, mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, starting during the first administration of the test item and lasting throughout the whole study period up to the day prior necropsy.
- Time schedule for arena observations: before the first administration of the test item and then once weekly throughout treatment.
- Clinical observations included: skin/fur, secretion/excretion, posture

BODY WEIGHT: Yes
- Time schedule for examinations: 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 post natal days (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 in Days 0, 4, 7, 11, 14, 17 and 29 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was expected or noted at visual inspection of the water bottles.

OTHER:
Haematology, coagulation, clinical chemistry, neuronal examination, thyroid hormone analysis (for details please refer to IUCLID section 7.5.1)

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 (pre-test period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in all male parental generations (P0): testis weight, epididymides weight, seminal vesicle weight, prostate weight, histopathological examination of testes to examine the tubular stages of the spermatogenic cycle (control and high dose group only and males that failed to sire).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was colleced, if possible.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight gain on PND 1, 4, 7 and 13, clinical signs, anogenital distance (AGD) on PND 1, presence of nipples/areolae in male pups on Day 13

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; the stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood was collected on PND 4 and PND 14 – 16, if possible. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation.
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: F1: two surplus pups per litter (if possible) on PND 4 and two surplus pups per litter on PND 14-16
- Parameters examined: T4, TSH
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals were sacrificed following completion of the mating period (minimum of 28 days after administration).
- Maternal animals: Females which delivered were exsanguinated on PND 14 – 16.
- Females which failed to deliver were sacrificed on post-coitum Day 25 or on Day 27 in case there was evidence of mating; without evidence of mating females were sacrificed 24 days after the last day of mating period.

GROSS NECROPSY
All animals were subjected to a full post mortem examination with special attention being paid to the reproductive organs. Any abnormalities were recorded. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation site were present, non-gravid uteri were stained (Salewski technique) in order to detect any former implantation sites. In addition, the number of corpora lutea was recorded.

ORGAN WEIGHTS
The following organs were weighed for all selected animals at necropsy: heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus, brain, cervix, epididymis, adrenal gland, coagulation gland (including seminal vesicles), parathyroid gland (together with the thyroid), prostate gland, seminal vesicles, thyroid gland. For all remaining animals (incl. females that failed to deliver pups) the following organs were weighed: epididymis, coagulation gland (including seminal vesicles), parathyroid gland (together with thyroid), prostate gland, seminal vesicles, thyroid gland and testes.

HISTOPATHOLOGY
Representative samples of the following tissues were collected from five male and five female animals per dose group and preserved in 10% neutral buffered formalin or modified Davidson’s fixative: aorta artery, nasopharynx body cavity, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal gland, coagulation gland, Harderian gland, lacrimal gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gut-associated lymphoid tissue, heart, kidney, all gross lesions/masses, large intestine (cecum, colon and rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord, spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus and vagina. Except for aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue, all tissues were embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin.
The following tissues were prepared for microscopic examination for all remaining animals (incl. males that failed to sire and females that failed to deliver pups): cervix, epididymis, coagulation gland, mammary gland, parathyroid gland (only collected if present in the routine section of the thyroid), pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, ovaries, testes, uterus, vagina, gross lesions/masses.
For details please refer to Table No. 1 under “Any other information on material and method incl. tables”.


Postmortem examinations (offspring):
SACRIFICE
- On PND 4, the surplus pups (> 8 per litter) were euthanized by decapitation. From two surplus pups per litter, blood was colleced, if possible. All remaining pups were sacrificed on PND 14 – 16 by i.p. injection of sodium pentobarbital (Euthasol 20%) except for the two pups per litter selected for blood collection.
- The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external examination. Sex was determined both externally and internally. All abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed
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:
Group 2 (15 mg/kg bw/day) vs. Group 1 (control)
Group 3 (50 mg/kg bw/day) vs. Group 1 (control)
Group 4 (150 mg/kg bw/day) vs. Group 1 (control)

Parametric analysis: 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 analysis: 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:
- 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
- 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
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 offspring on Day 4 before culling/Number of live offspring on Day 1 after littering) x 100
- Lactation index [%] = (Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No findings were noted during the weekly arena observations in this study. Salivation was observed after dosing in 7/10 females treated at 50 mg/kg bw/day between Days 13 - 18 and Days 30 - 51 and at 150 mg/kg bw/day in the majority of animals from the second week of treatment onwards. The effect was considered to be rather a physiological response thana sign of systemic toxicity, due to the nature and minor severity of the effect and its time of occurrence (after dosing). Any other clinical signs noted during the treatment period included hunched posture, piloerection, alopecia, scales and scabs. However, these occurred only in the control group or were within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions of the study and did not show any dose-related trend.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item-related mortality. 1/10 females administered 15 mg/kg bw/day died at blood sampling on the scheduled day of necropsy. The death was considered to be related to the blood sampling procedure and not related to the treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Females dosed at 150 mg/kg bw/day showed a significantly increased relative food consumption on Days 14 - 17 post-coitum. This effect was considered incidental and not related to treatment since no trend was apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related, statistically significant, decrease of 20% in reticulocytes was noted in males at 150 mg/kg bw/day when compared with concurrent controls. The mean value remained within historical control range. In females at 150 mg/kg bw/day, an increase of 45% and 10% was noted in neutrophils and lymphocytes (not statistically significant), respectively, when compared with concurrent controls. A concurrent increase of 19% was noted in white blood cells (WBC) for these females (not statistically significant). Mean values remained within historical control, however the individual WBC and lymphocyte values of 3/5 females were at or above the upper limit of historical control data. No other treatment-related changes were noted in haematology.
Coagulation parameters (prothrombin time (PT) and activated partial thromboplastine time (APTT) were considered unaffected by treatmenr up to 150 mg/kg bw/day. The apparent treatment-related decrease noted in mean prothrombin time (PT) for treated males was attributed to the relatively high mean control values and was therefore considered unrelated to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Findings in clinical biochemistry were only observed in animals treated at 150 mg/kg bw/day. In female animals, a 20% decreased urea concentration, a 11% lower concentration in creatinine and a 43% higher concentration of cholesterol was observed. All findings were statistically significant, but mean values remained in the range of historical control data. Creatinine values of single animals (3/5 females) were below the P5 value of the historical range. Cholesterol concentrations of 2/5 females were above the P5 value of the historical control. Any other statistically significant variations noted in clinical chemistry values were considered to be unrelated to treatment due to the lack of a dose-related response (potassium and calcium in males, and albumin and chloride in females).
Higher mean values (not statistically significant) of ALP, glucose and bile acids were noted in males at 150 mg/kg bw/day. This was mostly caused by relatively high values in one male (no. 33). For this male, the ALP and glucose values were above the P95-value of the historical control range, whereas the other males of the 150 mg/kg bw/day group had normal values (within historical control range). Therefore, the higher values of ALP, glucose and bile acids of male no. 33 were considered a change finding and not reflect an effect of the test item. for remaining parameters, the individual values of male no. 33 were within the range of concurrent control and therefore considered normal.
The apparent treatment.related increase noted in mean creatinine values of males was attributed to the relatively low mean concurrent control value and was therefore considered unrelated to treatment. The slightly lower glucose concentration noted in females at 150 m g/kg bw/day was mostly caused by one females (no. 72) with a value below the P5-value of the historical control range. As this concerned a single animal, the lower glucose value was considered incidental and not toxicologically relevant.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, females treated at 150 mg/kg bw/day exhibited a significantly decreased (13%) mean grip strength of the hind legs when compared with concurrent controls. Mean values remained within the historical control range. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength of the males and 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, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item related findings were observed in liver and kidneys of the 150 mg/kg bw/day males. The findings comprised hepatocellular hypertrophy at a minimal degree in 5/5 males administered 150 mg/kg bw/day. An increased severity of hyaline droplet accumulation (up to moderate) and tubular basophilia (up to moderate)was noted in 150 mg/kg bw/day males. Furthermore, granular casts (slight) were observed in one 150 mg/kg bw/day male (no. 32).For details please refer to Table No. 3 under “Any other information on results incl. tables”. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
1/10 males administered 15 mg/kg bw/day showed massive tubular atrophy of testes and a reduced luminal sperm in in the epididymides, which served as explanation for unsuccessful mating. As the abnormality was observed in a single animal of the low dose group, the effect was considered unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
Serum levels of T4 in F0 males were comparable to concurrent control values.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment. One control animal (No. 48) revealed an extended estrus and one female (No. 54) treated at 15 mg/kg bw/day exhibited an irregular cycle. Both females delivered a normal litter. Due to the absence of an dose-related effect and absence of apparent correlation to pregnancy status, the finding was not considered treatment related.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm parameters were not considered affected by treatment. One male treated at 15 mg/kg bw/day failed to sire, which was in line with histopathological findings (massive tubular atrophy of testes and a reduced luminal sperm in in the epididymides) at microscopical examination. Based on the single occurence in a male of the low dose group and the fact that these findings occasionally can be found in control males, these findings were considered to be unrelated to the test item.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index: The mating index was considered not to be affected by treatment. One of 10 females administered 15 mg/kg bw/day was unsuccessfully mated. The mating indices were 100% for the control, 50 and 150 mg/kg bw/day groups and 90% for the 15 mg/kg bw/day group.

Number of implantation sites: The number of implantation sites was considered not to be affected by treatment. 1/10 females dosed at 150 mg/kg bw/day had only three implantation sites, while the other 9 females treated at that dose had normal numbers of implantation sites. Therefore, the finding was considered incidental and unrelated to treatment.

Fertility index: The fertility index was not considered to be affected by treatment. 1/10 control animals, 1/10 females dosed at 15 mg/kg bw/day and 1/10 females administered 150 mg/kg bw/day were not pregnant. The effect was considered not to be treatment-related due to lacking histopathological changes in reproductive organs of these animals and due to the absence of a dose-response relationship.

Precoital time: Precoital time was considered to be unaffected by treatment, as the majority of females showed evidence of mating within three days.

Gestation index and duration: Gestation index and duration of gestation were considered not to be affected by treatment. Except for one control female (no. 46 which had implantation sites only) all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for all test item groups. This case of failed pregnancy occured without related histopathological changes in reproductive organs.

Parturition/Maternal care: No signs of difficult or prolonged parturition were noted amonf 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.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect observed
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
LOAEL
Remarks:
parental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occured among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment. One pup at 15 mg/kg bw/day (litter no. 52) showed a distended abdomen on PND 13. Based on this observation, this pup was sacrificed in extremis. Based on the incidental occurence in the low dose group, this clinical sign was considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Post-implantation survival index: 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 indices were 91% (control group), 91% (15 mg/kg bw/day), 87% (50 mg/kg bw/day) and 88% (150mg/kg bw/day), respectively.
Litter size: The mean number of living pups at first litter check was considered not to be affected by treatment. Live litter sizes were 12.9 (control group), 12.4 (15 mg/kg bw/day), 811.5 (50 mg/kg bw/day) and 10.9 (150 mg/kg bw/day), respectively.
Mean litter sizes at 50 and 150 mg/kg bw/day were lower compared with concurrent control (11.5 and 10.9 versus 12.9; mot statistically significant), which was explained by the small size of a few litters and a large control litter size compared to historical control data. Litter sizes at 50 and 150 mg/kg bw/day remained within normal limits and were not statistically significant. Therefore, the intergroup differences were not attributed to treatment.
Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not affected by treatment. Live birth indices were 100% (control group), 100% (15 mg/kg bw/day), 99% (50 mg/kg bw/day) and 98% (150mg/kg bw/day), respectively. One pup of the 50 mg/kg bw/day dose group and two pups of the 150 mg/kg bw/day dose group were found dead at first litter check. As the mortality incidence remained within the range considered normal for pups of this age, the effect was not considered toxicologically relevant.

Viability index: The number of live offspring on PND 4 before culling compared to the number of offspring on Day 1 was considered unaffected by treatment. Viability indices were 100% (control group), 98% (15 mg/kg bw/day), 100% (50 mg/kg bw/day) and 99% (150mg/kg bw/day), repsectively. Two pups at 15 mg/kg bw/day and one pup at 150 mg/kg bw/day went missing (presumably cannibalized) on PND 4 and 2, respectively. Since the mortality indices were not dose-related and remained within the range considered normal for pups of this age, there was no toxicological relevance attributed to these findings.

Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. The lactation indices were 100% (control group), 98% (15 mg/kg bw/day), 100% (50 mg/kg bw/day) and 100% (150mg/kg bw/day), respectively. One pup at 15 mg/kg bw/day was sacrificed in extremis on PND 13 due to a distended abdomen. The finding was considered not to be toxicologically relevant as there was no dose-response relationship and the mortality incidence remained within the range considered normal for pups of this age.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio: The sex ratio was considered not to be affected by treatment and remained within the historical control range. A few litters caused a statistically significant different sex ratio at 150 mg/kg bw/day. In the control group there were two litters with more females than males whereas at 150 mg/kg bw/day there were two litters with more males than females. The intergroup differences were attributed to biological variation and judged to be unrelated to treatment.

Anogenital distance: The mean anogenital distance was slightly increased (3.01 mm) in male pups at 150 mg/kg bw/day when compared to control animals (2.68 mm) (not statistically significant), but remained within the historical control data. Anogenital distance in female pups was not affected.
Areola/nipple retention: Treatment up to 150 mg/kg bw/day had no effect on areola/nipple retention, as none of the male pups revealed nipples on PND 13.
For details please refer to Table No. 13 under “Any other information on results incl. tables”.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic, treatment-related findings. The nature and incidence of abnormalities remained within the range considered normal for pups of this age and were not considered to be related to treatment. One pup at 15 mg/kg bw/day revealed a distended abdomen and was sacrificed on PND 13 in extremis. As only a single treated pup was affected and in the absence of the dose-related response, this finding was regarded as unrelated to treatment.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean T4 level in female pups was slightly increased (6.31 µg/dL, 8%) at 150 mg/kg bw/day when compared with concurrent controls (5.82 µg/dL). There was no statistical significance and the mean value remained within the range of historical control data. Thus, the difference was considered not toxicologically significant.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 2: Liver weights after treatment in percent

  Males Females
Dose level (mg/kg): 15 50 150 15 50 150
LIVER    
Absolute 0 4 9 5 3 14
Relative to body weight 2 5 15** 9 0 17*
*: P<0.05, **: P<0.01

Table No. 3: Histopathological findings in males

Males
Dose level (mg/kg bw) 0 15 50 150
Liver: Hepatocellular hypertrophy
minimal 0/5 0/5 0/5 5/5
Kidneys
Hyaline droplet accumulation
Minimal 4/5 3/5 5/5 1/5
Slight  1/5 1/5 0/5 2/5
Moderate 0/5 0/5 0/5 1/5
Tubular basophilia 
 Minimal 1/5 2/5 4/5 3/5
Slight  0/5 0/5 0/5 1/5
Moderate 0/5 0/5 0/5 1/5
Granular casts
Slight  0/5 0/5 0/5 1/5
Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was 50 mg/kg bw/day, based on alpha 2u-nephropathy in male rats at 150 mg/kg bw/day. The finding was is specific for male rats only and not relevant for humans.
The NOAEL for fertility and for development were both derived to be ≥ 150 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

N-Ethylcaprolactam was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD guideline 422 and in compliance with GLP (Charles River Laboratories, 2019).

Groups of 10 male and 10 female Wistar Han rats were exposed daily to the test item by oral gavage at doses of 15, 50 or 150 mg/kg bw/day. A similar constituted group of 10 mal and 10 female animals received the vehicle (water) and served as control. The dose levels for the main study were selected based on the results of a preliminary 10-days dose range finding study.

In the present study, male rats were treated for 29 days, beginning 14 days prior to mating. Females that delivered were treated for 50 – 55 days, starting two weeks prior to mating, during mating, during the duration of pregnancy and until 13 – 15 days after delivery. Females which failed to deliver were treated for 42 to 52 days.

Accuracy and homogeneity of the test item formulations were confirmed by analytical methods. The following parameters were evaluated in the study: Mortality/moribundity, clinical signs, functional observations, body weight, food consumption, estrous cycle determination, clinical pathology, thyroid (T4) hormone levels in females, gross necropsy findings, organ weights and histopathological examinations. In addition, the following parameters on reproduction were examined: Mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition and maternal care.

For details on parental systemic toxicity (mortality, clinical signs, functional observations, haematology, clinical chemistry, organ weights, gross pathology and histopathological findings) please refer to Section 7.5 “Repeated dose toxicity”.

There was no reproductive toxicity observed up to the highest tested concentration. Length and regularity of the estrous cycle were not considered to have been affected by treatment. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any induction for abnormal spermatogenesis. Mating and fertility index, the number of implantation sites and pre-coital time were not altered by treatment.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL for reproductive toxicity was derived to be ≥150 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Oral (OECD 422), rat: NOAEL developmental > 150 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

N-Ethylcaprolactam was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD guideline 422 and in compliance with GLP (Charles River Laboratories, 2019).

Groups of 10 male and 10 female Wistar Han rats were exposed daily to the test item by oral gavage at doses of 15, 50 or 150 mg/kg bw/day. A similar constituted group of 10 mal and 10 female animals received the vehicle (water) and served as control. The dose levels for the main study were selected based on the results of a preliminary 10-days dose range finding study.

In the present study, male rats were treated for 29 days, beginning 14 days prior to mating. Females that delivered were treated for 50 – 55 days, starting two weeks prior to mating, during mating, during pregnancy and until 13 – 15 days after delivery. Females which failed to deliver were treated for 42 to 52 days.

Accuracy and homogeneity of the test item formulations were confirmed by analytical methods. The following parameters were evaluated in the study: Mortality/moribundity, clinical signs, functional observations, body weight, food consumption, estrous cycle determination, clinical pathology, thyroid (T4) hormone levels in females, gross necropsy findings, organ weights and histopathological examinations. In addition, the following parameters on development were examined: 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)).

For details on parental systemic toxicity (mortality, clinical signs, functional observations, haematology, clinical chemistry, organ weights, gross pathology and histopathological findings) please refer to Section 7.5 “Repeated dose toxicity”.

There were no effects on developmental toxicity related to treatment up to the highest dose level tested. Gestation index and duration of gestation were considered not to be affected by treatment. 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. Post-implantation survival index, litter sizes, live birth index, viability index and lactation index were considered unaffected by treatment.

The clinical signs observed incidentally remained within the range considered normal for pups of thus are and were therefore considered to be unrelated to treatment. Body weights, sex ratio, serum T4 levels were considered not to be affected by treatment. Anogenital distance in pups remained within historical control data. Treatment up to 150 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. No macroscopic findings were noted among pups that were considered to be treatment-related.

In conclusion, the NOAEL for development was derived to be ≥ 150 mg/kg bw/day. 

Justification for classification or non-classification

The available data on reproduction and developmental toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.