Registration Dossier

Administrative data

Description of key information

Oral (OECD 422), male rat: NOAEL = 50 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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 oral 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
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
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.
Observations and examinations performed and frequency:
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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of all F0 animals was collected on the day of necropsy from retro-oribital sinus. Blood of F1 animals was collected on PND4 at culling (decapitation of surplus animals) from two surplus pups per litter (if available from 1 male and 1 female pup) and on PND 14 – 16 from two pups per litter by aorta puncture.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males were fasted overnight before sampling, females were not fasted.
- How many animals: 5 animals per group
- Parameters checked: white blood cells, neutrophils (absolute), lymphocytes (absolute), monocytes (absolute), eosinophils (absolute), basophils (absolute), red blood cells, reticulocytes (absolute), red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of all F0 animals was collected on the day of necropsy from retro-oribital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males were fasted overnight before sampling, females were not fasted.
- How many animals: 5 animals per group
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during Week 4 of treatment and females during the last week of lactation (PND 10-12)
- Dose groups that were examined: all dose groups, 5 selected animals per dose group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind limb grip strength and locomotor activity

IMMUNOLOGY: No

THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood of all F0 animals was collected on the day of necropsy from retro-oribital sinus.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males were fasted overnight before sampling, females were not fasted.
- How many animals: 5 animals per group
- Parameters examined: total thyroxine (T4) and thyroid stimulating hormone (TSH)
Sacrifice and pathology:
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”.
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.
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.
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
Description (incidence and severity):
Significantly increased relative liver weight (15% and 17%) was found in male and female animals administered 150 mg/kg bw/day. In males the microscopic correlate for this weight increase was hepatocellular hypertrophy. In females, there was no microscopic correlate for the increase in liver weight. For one female (no. 77) the increased hepatic weight correlated to the macroscopically observed enlargement of the liver. The relative mean thymus weight of males treated at 150 mg/kg bw/day was 21% increased when compared with concurrent controls (not statistically significant). This effect was related to one single animal, thus the higher thymus weight was considered incidental and not toxicologically relevant. For details please refer to Table No. 2 under “Any other information on results incl. tables”.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Liver enlargement was observed in 1/10 females (No. 68) treated at 50 mg/kg bw/day and in 1/10 females (No. 77) treated at 150 mg/kg bw/day. At 50 mg/kg bw/day, the individual liver weight of female No. 68 and the mean liver weight of females at 50 mg/kg bw/day (absolute and relative) were within normal limits and comparable to the liver weights of the control group. Furthermore, there was no microscopic correlate and therefore this finding was regarded to be unrelated to treatment with the test item. At 150 mg/kg bw/day, there was a statistically significant increase in mean relative liver weight and therefore this macroscopic finding of female no. 77 (150 mg/kg bw/day) was considered possible test-item related. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
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.
Key result
Dose descriptor:
LOAEL
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
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
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
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

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-globulin nephropathy observed in male rats at 150 mg/kg bw/day. Alpha 2u-globulin nephropathy is specific for male rats only and not relevant for humans.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 1), and are thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral repeated dose toxicity:

The test item was investigated 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 male 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, the duration of pregnancy and until 13 – 15 days after delivery. Females which failed to deliver were treated for 42 to 52 days.

Observations and examinations of the animals included mortality, clinical signs, functional observations (for 5 selected animals/sex/group), body weight, food consumption, estrous cycle determination, haematology (for 5 selected animals/sex/group), clinical chemistry (for 5 selected animals/sex/group), measurement of thyroid hormone T4, gross necropsy, organ weights and histopathology.

Accuracy and homogeneity of the test item formulations in water were confirmed by analytical methods. There was no treatment related mortality observed up to the highest tested dose of 150 mg/kg bw/day and food consumption and body weight gain remained as well unaffected by treatment. Clinical signs of toxicity comprised salivation in 7/10 female rats observed at 50 mg/kg bw/day and in the majority of animals at 150 mg/kg bw/day. The effect was considered to be a physiological response rather than a sign of systemic toxicity.

Functional tests revealed a decreased mean grip strength of hind legs (-13%) in females in the high dose group when compared with concurrent controls. However, the effect was not considered toxicologically relevant as mean values remained within normal limits and no corroborative clinical signs in the arena observations or anatomic pathology alterations were observed.

Hematological analysis showed a statistically significant decrease (-20%) in reticulocytes in males in the 150 mg/kg bw/day dose group and a non-significant increase in white blood cells (+19%), neutrophil (+45%) and lymphocyte (+10%) concentration in females administered 150 mg/kg bw/day when compared with concurrent controls. The findings were regarded as non-adverse, as the values remained within the range considered normal for rats of this strain and age and due to the absence of adverse anatomic pathology alterations.

Differences in clinical chemistry parameters from control animals comprised a statistically significant reduced urea (-20%) and creatinine (-11%) concentration and a significantly increased cholesterol level (+43%) in female rats of the high dose group, but values remained within those of the historical control range. These findings in clinical pathology were regarded as non-adverse, as the values remained within the range considered normal for rats of this strain and age and due to the absence of adverse anatomic pathology alterations.

Increased relative liver weight was observed in males (+15%) and females (+17%) administered 150 mg/kg bw/day. Microscopically, this correlated with minimal hepatocellular hypertrophy in males (there was no microscopic correlation in females). The hepatic effects were considered non-adverse, there were no degenerative, inflammatory or proliferative findings and no corroborative changes in any of the clinical chemistry parameters. In addition, there was a 21% increase in the relative mean thymus weight (non-significant) of males in the high dose group when compared with concurrent control. All values, except for one male (no. 32) remained within historical control data. As this concerned a single animal, the higher thymus weight as considered incidental and not toxicologically relevant. Moreover, the increase in relative thymus weight was not accompanied by any anatomic pathology alterations.

Histopathology revealed test item-related findings in kidneys comprising hyaline droplet accumulation and tubular basophilia as well as granular casts in males of the high dose group. The combination of findings is known as alpha 2u-globulin nephropathy and was considered as adverse. However, it should be taken into account that hyaline droplets are considered to represent alpha 2u-globulin, a normal protein present in male rats and absent in humans.

In conclusion, based on the alpha 2u-globulin nephropathy observed in male rats treated at 150 mg/kg bw/day, a NOAEL (systemic) of 50 mg/kg bw/day was derived.

Justification for classification or non-classification

Histopathological abnormalities were observed in male rats treated at 150 mg/kg bw/day in an OECD 422 subacute toxicity study. The adverse effects were identified as alpha 2u-globulin nephropathy. Based on these effects, the Lowest Observed Effect Level (LOAEL) was established as 150 mg/kg bw/day and a NOAEL of 50 mg/kg bw/day was derived.

According to the criteria of Regulation (EC) No. 1272/2008 (CLP), Annex I, Section 3.9, a LOAEL guidance value of ≤ 100 mg/kg bw/day based on data obtained in a sub-chronic (90-day) is used to indicate the necessity for classification as specific target organ toxicant after repeated exposure. If data obtained in a study of shorter duration are available and used for hazard assessment, the guidance value must be modified, taking the difference in exposure periods into account. The ECHA Guidance on the Application of the CLP Criteria (version 5.0, July 2017), therefore, stipulates an equivalent guidance value for a sub-acute (28-day) study of ≤ 300 mg/kg bw/day. The conversion of LOAEL guidance values is done by multiplying the original guidance value with a factor of 3, approximating the ratio of the exposure periods (90 / 28 = 3.2; approx. 3). As the findings were observed at a dose level of 150 mg/kg bw/day, a classification of the test substance may be considered.

However, alpha 2u globulin-nephropathy is a finding specific for male rats and considered not relevant for humans, as humans do not have a functional alpha 2u-globulin gene (IARC, 1999). Thus, the available data on repeated dose toxicity following oral exposure for 28 days (males) according to OECD guideline 422, N-ethylcaprolactam is not considered to meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) for specific target organ toxicity after repeated exposure. The data are therefore conclusive but not sufficient for classification.

Reference: IARC (1999) Species Differences in Thyroid, Kidney and Urinary Bladder Carcinogenesis. IARC Scientific Publications No. 147 (CC Capen, E Dybing, JM Rice and JD Wilbourn Eds.), 225 pp.