2-hydroxy-3-(prop-2-enoyloxy)propyl 2-methyl-2-propylhexanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 17, 2016 to August 01, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 17, 2016 to August 01, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Deutschland, Sulzfeld, Germany.
Age at start: approximately 10-12 weeks.
Number of animals: 40 females and 40 males.
Acclimatization: at least 5 days prior to start of pretest (females) or treatment (males).
Temperature: 18 to 24°C
Relative humidity: 40 to 70%
Light period: 12-hour light/12-hour dark cycle.
Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
Water: free access to tap-water

Route of administration:

oral: gavage

Details on route of administration:

Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. No correction was made for the purity/composition of the test substance. Appearance of formulations: clear solution.

Vehicle:

propylene glycol

Details on oral exposure:

The test substance, formulated in propylene glycol, was administered daily by oral gavage using a plastic feeding tube to SPF bred Wistar Han rats. Formulations were placed on a magnetic stirrer during dosing. Dose volume: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Remarks:

UPLC system

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (samples of 16 June 2016) according to a validated method (Test Facility Study No. 512422). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 41 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (one control group and three treated groups were tested)

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a 14-day dose range finding study (Test Facility Study No. 512419; see APPENDIX 5), the dose levels for this combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw. The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF bred Wistar Han rats. This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 41 days. This study covered a 28-day repeated dose toxicity study with a reproduction/developmental toxicity screening.

Positive control:

-

Observations and examinations performed and frequency:

- Mortality / Viability: At least twice daily.
- Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least immediately (0-30 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
- Functional Observations: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex (score 0 = normal/present, score 1 = abnormal/absent), and fore- and hind-limb grip strength, recorded as the mean of three measurements per animal, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system)). Total movements and ambulations are also reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (including arena observation, if applicable) and were started immediately (0-30 min) after dosing.
- Body weights: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
1. Haematology: The haematology parameters (white and red blood cells, reticulocytes, platelets, haemoglobin and haematocrit) were determined in blood prepared with K3-EDTA as an anti-coagulant. The clotting parameters (PT, APTT) were determined in plasma prepared with citrate as anticoagulant.
2. Clinical Biochemistry: The clinical biochemistry parameters (enzymes, ions, total protein, albumin, bile acids, bilirubin, urea, creatinine, glucose, and cholsterol) were determined. All parameters were determined in plasma, except for bile acids which were determined in serum.

Sacrifice and pathology:

- Termination: All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non selected females were not deprived of food. All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. Necropsy was then conducted (spontaneous deaths were also nalysed).
- Macroscopic examination: After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (selected 5 animals/sex/group and all animals that died spontaneously): ovaries adrenal glands, pancreas, aorta, jejunum, ileum, brain (cerebellum, midbrain, cortex), pituitary gland, caecum, preputial gland, rectum, colon, salivary glands (mandibular, sublingual), sciatic nerve, duodenum, seminal vesicles, skeletal muscle, eyes (with optic nerve), Harderian gland, skin, spinal cord (cervical, midthoracic, lumbar), female mammary gland area, spleen, femur including joint, sternum with bone marrow, heart, stomach, ileum, testes, thymus, kidneys, thyroid including parathyroid, lacrimal gland, tongue, larynx, trachea, liver, urinary bladder, lung, uterus, lymph nodes (mandibular, mesenteric), vagina, nasopharynx. esophagus, cervix, preputial gland, clitoral gland, prostate gland, coagulation glands, seminal vesicles, epididymides
- Organ weights: On the scheduled day of necropsy, terminal body weight was recorded from all males and the selected 5 females/sex/group. The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy (selected 5 animals/sex/group): adrenal glands, prostate, brain, seminal vesicles including coagulating glands, epididymides, spleen, heart, testes, kidneys; thymus, liver, thyroid (including parathyroid if detectable), ovaries, uterus (including cervix). Absolute organ weights and organ to body weight ratios were reported.
- Histotechnology: All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. (The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin).
- Histopathology: The following slides were examined by a pathologist (from preserved organs and tissues): testes, thyroid gland, stomach, liver and kidneys.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during the observation period. Rales were noted for 1-3 days for one animal/sex at 100 mg/kg bw and for two animals/sex at 300 and 1000 mg/kg bw. At it only concerned a couple of animals for a limited number of days it was not considered toxicologically relevant. Salivation seen after dosing among several animals at 100 mg/kg bw and all animals at 300 and 1000 mg/kg bw was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance. Incidental findings that were noted included hunched posture and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female in the 100 mg/kg bw dose group was found dead on Day 17 post-coitum. There were no clinical signs recorded or changes seen on her bodyweight or food consumption before death. At necropsy, beginning autolysis and in the uterus 12 normal fetuses and 2 early resorptions were recorded. No abnormalities of these fetuses were observed externally. At microscopic examination, massive ulceration of the trachea was noted, that most likely was caused by the gavage procedure and was considered to be the cause of death. This death was therefore considered to be unrelated to the test substance.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw. The statistically significantly increased body weight gain noted at 1000 mg/kg bw on Day 4 of lactation was not considered toxicologically relevant as it concerned a slight increase for one occasion only and all values were within normal limits.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. Absolute and relative food consumption were slightly reduced during the first week of treatment at 300 and 1000 mg/kg bw for males and/or females. As this decrease was minimal and recovered to control values during the remainder of the treatment period, it was not considered toxicologically relevant.

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were considered to be unaffected by treatment. The statistically significant changes of mean corpuscular volume in females at 100 and 1000 mg/kg bw were considered not to be toxicologically relevant as they occurred in the absence of a dose-related distribution, no corroborative haematological findings were noted and values remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 1000 mg/kg bw, creatinine and inorganic phosphate values were significantly and dose dependently increased in males. In addition, cholesterol concentrations were increased for females at 1000 mg/kg bw. Any remaining statistically significant changes noted for males at 100 and 300 mg/kg bw were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher liver weights (absolute and relative to body weights) were noted in the 1000 mg/kg bw group males (relative weights 29% higher than control) and females (relative weights 22% higher than control) and test substance-related higher kidney weights (relative to body weights) were noted in the 1000 mg/kg bw group males (15% higher than control). Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy revealed alterations in the stomach, liver and kidney that were considered to have arisen as a result of treatment. Kidney abnormalities were noted for males of all treated groups, and consisted of:
- Enlarged kidneys in 1/10 males at 300 mg/kg bw, and 5/10 males at 1000 mg/kg bw, accentuated lobular pattern in 5/10 males at 1000 mg/kg bw, pale discolouration in 2/10 males at 100 mg/kg bw, 1/10 males at 300 mg/kg bw, and 8/10 males at 1000 mg/kg bw, flaccid kidneys in 1/10 males at 100 mg/kg bw, 2/10 males at 300 mg/kg bw, and 2/10 males at 1000 mg/kg bw.
- Additionally, irregular surface of the forestomach in 2/10 males, many red-brown foci in the forestomach in 1/10 males, an enlarged liver in 1/10 males, and red-brown discoloration of the liver in 1/10 males were noted at 1000 mg/kg bw.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings after treatment with the test substance were noted in both sexes in the liver and in the thyroid gland, stomach and kidneys in males. Centrilobular hepatocellular hypertrophy was recorded in the liver of a single male and female at 1000 mg/kg bw. A slightly increased incidence and severity of follicular cell hypertrophy in the thyroid gland, up to a slight degree, was present in two males each at 300 and 1000 mg/kg bw. Test substance-related microscopic findings were present in the forestomach in 2/5 males at 1000 mg/kg bw consisting of a combination of lymphogranulocytic inflammation, squamous hyperplasia and/or ulcer. An increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys starting at the dose level of 100 mg/kg bw and above.
At 1000 mg/kg, the following microscopic changes of the kidney were observed in males: an increased incidence and severity of tubular basophilia up to marked degree in all males, granular casts in 9/10 males, degeneration/dilation tubule in 6/10 males. At 300 mg/kg bw, degeneration/dilation tubule in the kidneys, at a minimal degree, was also recorded in 2/10 males. The remaining histologic changes were considered to be incidental findings

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Functional observations: No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: due to microscopic findings in the stomach (at 1000 mg/kg) and kidneys (at 300 and 1000 mg/kg)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

yes

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Analysis of dose preparations:

- Accuracy of preparation

The concentrations analysed in the different formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the control formulation.

- Homogeneity

The formulations of the groups 100 and 1000 mg/kg bw were homogeneous (i.e. coefficient of variation ≤ 10%).

- Stability

Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours.

Treatment up to 1000 mg/kg was well-tolerated; no mortality related to the treatment with the test item occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights, food consumptions and haematological parameters were noted. At 300 and 1000 mg/kg, test item-related macroscopic findings were present in male rats in the stomach, liver and kidney and test item-related microscopic findings were present in the liver of both sexes and thyroid gland, stomach and kidneys of male rats. In the thyroid gland a slightly increased incidence and severity of follicular cell hypertrophy was present in males treated at 300 and 1000 mg/kg. Follicular cell hypertrophy of the thyroid gland in rats is usually an adaptive response to induction of hepatic enzymes. This results in increase in the hepatic/biliary clearance of T3/T4 leading to increase in TSH and compensatory follicular cell hypertrophy and/or hyperplasia (Ref. 6). The minor increase in incidence and/or severity (up to slight degree) of follicular cell hypertrophy was regarded to be an adaptive change and considered to be nonadverse at the incidences and severities recorded (Ref. 7). A combination of lymphogranulocytic inflammation, squamous hyperplasia and/or ulcer was present in the forestomach of a few males treated at 1000 mg/kg. The macroscopic correlate of this finding was irregular surface and many red-brown foci in the forestomach recorded at necropsy. The findings recorded in the stomach of some male rats at 1000 mg/kg were considered to be most likely a local response to exposure with the test item, whereby the moderate ulceration of the forestomach can be regarded as adverse. There were test item-related organ weight changes seen in the liver of both sexes. Statistically significantly higher organ weights were recorded in males (absolute and relative to body weights increased by 26 and 28 %, respectively) and in females (absolute and relative to body weights increased by 20 and 22 %, respectively) at 1000 mg/kg. The microscopic correlate to the increased liver weight was centrilobular hepatocellular hypertrophy in a single female at 1000 mg/kg (for males no microscopic correlate was found). In the high dose group females the plasma cholesterol level was statistically significantly increased by 41 % when compared to concurrent controls. At microscopic examination, centrilobular hepatocellular hypertrophy at a minimal degree was recorded in the liver of a single male and female at 1000 mg/kg. The macroscopic correlate of this finding was discolored liver. In the absence of any degenerative findings in the liver, the minimal hepatocellular hypertrophy of the liver was considered to be a non-adverse finding (Ref. 8). At 300 and 1000 mg/kg, test item related statistical significant and dose dependent increase of creatinine and inorganic phosphate concentrations were noted in the blood plasma of male rats. At 1000 mg/kg the relative kidney weights were also increased in males. The microscopic correlate to the increased kidney weight in males was hyaline droplet accumulation and degeneration/dilation tubule. The hyaline droplet accumulation in male kidneys likely represents alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by degeneration/dilation tubule, formation of granular casts and tubular basophilia.

This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 9). In addition, at 1000 mg/kg, hyaline droplet accumulation was accompanied by an increased incidence and severity of tubular basophilia and granular casts in the kidneys. The degeneration/dilation tubule, formation of granular casts and tubular basophilia were considered to be secondary to the hyaline droplet accumulation and were considered adverse in male rats at 300 and 1000 mg/kg since they represent degenerative changes. Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). Developmental results: No adverse developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). At 1000 mg/kg, the pup weights were lower when compared to concurrent controls. This change differed up to 8% and was not statistical significant when compared to concurrent controls, therefore it was considered to be non-adverse. Conclusion Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived: Parental NOAEL males: 100 mg/kg due to microscopic findings in the stomach (at 1000 mg/kg) and kidneys (at 300 and 1000 mg/kg) Parental NOAEL females: at least 1000 mg/kg Reproduction NOAEL: at least 1000 mg/kg Developmental NOAEL: at least 1000 mg/kg

Conclusions:

Under the study conditions, the oral 28 d NOAEL values were determined at 100 mg/kg bw in male rats due to microscopic findings in the stomach (at 1000 mg/kg bw/day) and kidneys (at 300 and 1000 mg/kg bw/day) and at least 1000 mg/kg bw/day in female rats.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance according to OECD Guideline 422 and 407, EU Method B.7, OPPTS 870.3650 and 870.3050, in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females that delivered were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 5-7 days of lactation (for 41-47 days). Females that failed to deliver healthy offspring were exposed for 41 days. Formulations were analyzed once during the study to assess accuracy and homogeneity and stability. The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 h at room temperature protected from light. Treatment up to 1000 mg/kg bw was well-tolerated; no mortality related to the treatment with the test substance occurred, no treatment-related changes during functional observation tests were seen, and no toxicologically relevant clinical signs or changes in body weights, food consumptions and haematological parameters were noted. At 300 and 1000 mg/kg bw, test substance-related macroscopic findings were present in male rats in the stomach, liver and kidney and test substance-related microscopic findings were present in the liver of both sexes and thyroid gland, stomach and kidneys of male rats. In the thyroid gland a slightly increased incidence and severity of follicular cell hypertrophy was present in males treated at 300 and 1000 mg/kg bw. Follicular cell hypertrophy of the thyroid gland in rats is usually an adaptive response to induction of hepatic enzymes. This results in increase in the hepatic/biliary clearance of T3/T4 leading to increase in TSH and compensatory follicular cell hypertrophy and/or hyperplasia. The minor increase in incidence and/or severity (up to slight degree) of follicular cell hypertrophy was regarded to be an adaptive change and considered to be nonadverse at the incidences and severities recorded. A combination of lymphogranulocytic inflammation, squamous hyperplasia and/or ulcer was present in the forestomach of a few males treated at 1000 mg/kg bw. The macroscopic correlate of this finding was irregular surface and many red-brown foci in the forestomach recorded at necropsy. The findings recorded in the stomach of some male rats at 1000 mg/kg bw were considered to be most likely a local response to exposure with the test substance, whereby the moderate ulceration of the forestomach can be regarded as adverse. There were test substance-related organ weight changes seen in the liver of both sexes. Statistically significantly higher organ weights were recorded in males (absolute and relative to body weights increased by 26 and 28%, respectively) and in females (absolute and relative to body weights increased by 20 and 22%, respectively) at 1000 mg/kg bw. The microscopic correlate to the increased liver weight was centrilobular hepatocellular hypertrophy in a single female at 1000 mg/kg bw (for males no microscopic correlate was found). In the high dose group females the plasma cholesterol level was statistically significantly increased by 41% when compared to concurrent controls. At microscopic examination, centrilobular hepatocellular hypertrophy at a minimal degree was recorded in the liver of a single male and female at 1000 mg/kg bw. The macroscopic correlate of this finding was discolored liver. In the absence of any degenerative findings in the liver, the minimal hepatocellular hypertrophy of the liver was considered to be a non-adverse finding. At 300 and 1000 mg/kg bw, test substance-related statistical significant and dose dependent increase of creatinine and inorganic phosphate concentrations were noted in the blood plasma of male rats. At 1000 mg/kg bw the relative kidney weights were also increased in males. The microscopic correlate to the increased kidney weight in males was hyaline droplet accumulation and degeneration/dilation tubule. The hyaline droplet accumulation in male kidneys likely represents alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by degeneration/dilation tubule, formation of granular casts and tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. In addition, at 1000 mg/kg bw, hyaline droplet accumulation was accompanied by an increased incidence and severity of tubular basophilia and granular casts in the kidneys. The degeneration/dilation tubule, formation of granular casts and tubular basophilia were considered to be secondary to the hyaline droplet accumulation and were considered adverse in male rats at 300 and 1000 mg/kg bw since they represent degenerative changes. Under the study conditions, the oral 28d NOAEL values were determined at 100 mg/kg bw in male rats (due to microscopic findings in the stomach at 1000 mg/kg bw/day) and kidneys at 300 and 1000 mg/kg bw and at least 1000 mg/kg bw in female rats (de Raaf-Beekhuijzen, 2016).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

GLP compliance:

yes

Limit test:

no

Justification for study design:

Repeated dose toxicity combined study for the assessment of substance-related systemic effects and impact on the reproduction period.

Test material

Test material information

Constituent 1

Reference substance name:

Neodecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Molecular formula:

C16H28O5

IUPAC Name:

Neodecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Constituent 2

Reference substance name:

Neo undecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Molecular formula:

C17H30O5

IUPAC Name:

Neo undecanoic acid, 1-ester with 1,2,3-propanetriol mono-2-propenoate

Constituent 3

Reference substance name:

glycerol 1,3-dineodecanoate

Molecular formula:

C23H44O5

IUPAC Name:

glycerol 1,3-dineodecanoate

Constituent 4

Reference substance name:

Sum of other constituents, each of them at <1%

Molecular formula:

Not available

IUPAC Name:

Sum of other constituents, each of them at <1%

Test material form:

liquid

Specific details on test material used for the study:

Batch no.: JBGJ0045R
Purity: 100% (UVCB)
Appearance: clear yellowish liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Deutschland, Sulzfeld, Germany.
Age at start: approximately 10-12 weeks.
Number of animals: 40 females and 40 males.
Acclimatization: at least 5 days prior to start of pretest (females) or treatment (males).
Temperature: 18 to 24°C
Relative humidity: 40 to 70%
Light period: 12-hour light/12-hour dark cycle.
Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
Water: free access to tap-water

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. No correction was made for the purity/composition of the test substance. Appearance of formulations: clear solution.

Details on mating procedure:

Mating procedures Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of 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 was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Detection of mating was not confirmed for animal no. 60 which did deliver live offspring. 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. Parturition The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed. Number of pups 417 pups. Identification of pups On PND 1, all pups were individually identified by means of subcutaneous injection of Indian ink.

Analytical verification of doses or concentrations:

yes

Remarks:

UPLC system

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (samples of 16 June 2016) according to a validated method (Test Facility Study No. 512422). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 41 days.
(Pups were not dosed directly but could have potentially been exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/feces).

Frequency of treatment:

Once daily

Details on study schedule:

7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

10 (one control group and three treated groups were tested)

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a 14-day dose range finding study (Test Facility Study No. 512419; see APPENDIX 5), the dose levels for this combined 28-day repeated dose toxicity study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg bw. The test substance, formulated in propylene glycol, was administered daily by oral gavage to SPF bred Wistar Han rats. This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were exposed for 41 days. This study covered a 28-day repeated dose toxicity study with a reproduction/developmental toxicity screening.

Positive control:

-

Examinations

Parental animals: Observations and examinations:

- Mortality / Viability: At least twice daily.
- Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least immediately (0-30 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
- Functional Observations: The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex (score 0 = normal/present, score 1 = abnormal/absent), and fore- and hind-limb grip strength, recorded as the mean of three measurements per animal, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system)). Total movements and ambulations are also reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (including arena observation, if applicable) and were started immediately (0-30 min) after dosing.
- Body weights: Males and females were weighed on the first day of exposure (prior to first exposure) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.
- Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
1. Haematology: The haematology parameters (white and red blood cells, reticulocytes, platelets, haemoglobin and haematocrit) were determined in blood prepared with K3-EDTA as an anti-coagulant. The clotting parameters (PT, APTT) were determined in plasma prepared with citrate as anticoagulant.
2. Clinical Biochemistry: The clinical biochemistry parameters (enzymes, ions, total protein, albumin, bile acids, bilirubin, urea, creatinine, glucose, and cholsterol) were determined. All parameters were determined in plasma, except for bile acids which were determined in serum.

Oestrous cyclicity (parental animals):

Macroscopic evaluation: Ovaries, uterus, vagina.
Weight: Ovaries, uterus (including cervix).
The number of former implantation sites and corpora lutea were recorded for all paired females.
Reproductive parameters: cfr "reproductive indices"

Sperm parameters (parental animals):

Macroscopic examination: Prostate gland, testes, seminal vesicles, epididymides.
Weight: Prostate gland, testes, seminal vesicles including coagulating glands, epididymides.
Histophatology: Additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.

Litter observations:

Each litter was examined to determine the following:
- Mortality / Viability. The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups will be weighed on Days 1 and 4 of lactation.
- Sex: Determined for all pups on PND 1 and 4.

Postmortem examinations (parental animals):

- Termination: All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non selected females were not deprived of food. All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. Necropsy was then conducted (spontaneous deaths were also nalysed).
- Macroscopic examination: After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (selected 5 animals/sex/group and all animals that died spontaneously): ovaries adrenal glands, pancreas, aorta, jejunum, ileum, brain (cerebellum, midbrain, cortex), pituitary gland, caecum, preputial gland, rectum, colon, salivary glands (mandibular, sublingual), sciatic nerve, duodenum, skeletal muscle, eyes (with optic nerve), Harderian gland, skin, spinal cord (cervical, midthoracic, lumbar), female mammary gland area, spleen, femur including joint, sternum with bone marrow, heart, stomach, ileum, testes, thymus, kidneys, thyroid including parathyroid, lacrimal gland, tongue, larynx, trachea, liver, urinary bladder, lung, uterus, lymph nodes (mandibular, mesenteric), vagina, nasopharynx. esophagus, cervix, preputial gland, clitoral gland, prostate gland, coagulation glands, seminal vesicles, epididymides
- Organ weights: On the scheduled day of necropsy, terminal body weight was recorded from all males and the selected 5 females/sex/group. The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy (selected 5 animals/sex/group): adrenal glands, prostate, brain, seminal vesicles including coagulating glands, epididymides, spleen, heart, testes, kidneys; thymus, liver, thyroid (including parathyroid if detectable), ovaries, uterus (including cervix). Absolute organ weights and organ to body weight ratios were reported.
- Histotechnology: All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. (The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin).
- Histopathology: The following slides were examined by a pathologist (from preserved organs and tissues): testes, thyroid gland, stomach, liver and kidneys.
NB. The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups were analyzed and included: the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Postmortem examinations (offspring):

F1-generation pups:
- Termination: Pups surviving to planned termination were killed by decapitation between Days 5-7 of lactation. (Pup 9 of litter 66 was found dead during the weekend, and was fixed in an identified container containing 70% ethanol as it was not necropsied on the same day.)
- Macroscopic examination: All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

- Mating index (%), fertility index (%), conception index (%), gestation index (%) and duration of gestation.
- General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Offspring viability indices:

Percentage live males at first litter check, percentage live females at first litter check, percentage of postnatal loss and viability index (%).

Results and discussion

Results: P0 (first parental generation)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters and no morphological findings in the reproductive organs were noted.
- Mating index was not considered to be affected by treatment. All females showed evidence of mating.
- Fertility and conception index were not considered to be affected by treatment. A single female at 1000 mg/kg bw was not pregnant. Since this single occurrence was within normal limits and no other reproductive finding was noted in the high dose group, this was not considered to be related to treatment.
- Precoital time was considered to be unaffected by treatment.
- Number of implantation sites was considered to be unaffected by treatment. No toxicologically relevant effects on developmental parameters were noted. One pregnant female at 1000 mg/kg bw had implantation sites only. All the other pregnant females surviving until necropsy delivered live offspring.
- Gestation index and duration of gestation were considered to be unaffected 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.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on reproductive parameters and morphological findings in the reproductive organs were noted. Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2/10 couples at 1000 mg/kg bw with no offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. There were no morphological findings in the reproductive organs of neither sexes which could be attributed to the test substance. Spermatogenic staging profiles were normal for all males examined.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Treatment related:

no

Dose response relationship:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. Incidental clinical symptoms of pups consisted of lean appearance, red spots on head, blue snout, blue spot on the back, scabs on abdomen and no milk in the stomach. For the missing control pup on Day 2 of lactation, a wound at the left axillary region and less milk at first litter check were recorded. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. One pup in the control group was missing on Day 2 of Lactation. Three pups at 300 mg/kg bw and one pup at 1000 mg/kg bw were found dead at first litter check. The missing pup was most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups at 1000 mg/kg bw were slightly reduced on Days 1 and 4 of lactation when compared to the concurrent control group (i.e. 94% on Day 1 and 92% on Day 4 of lactation of the concurrent control values of body weights combined for both sexes).

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:

no effects observed

Description (incidence and severity):

Sex ratio was not considered to be affected by treatment.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. Lean appearance was recorded for 1 pup in group 1000 mg/kg bw and no milk in stomach was noted for 1 pup in group 1000 mg/kg bw. In group 300 mg/kg bw, scabs on abdomen was recorded for 1 pup. These findings were considered to be incidental.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Treatment related:

no

Dose response relationship:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Analysis of dose preparations:

- Accuracy of preparation

The concentrations analysed in the different formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test substance was detected in the control formulation.

- Homogeneity

The formulations of the groups 100 and 1000 mg/kg bw were homogeneous (i.e. coefficient of variation ≤ 10%).

- Stability

Formulations at the entire range were stable when stored at room temperature protected from light for at least 6 hours.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for reproduction and developmental toxicity in rats were determined to be at least 1000 mg/kg bw.

Executive summary:

A screening study was conducted to determine the reproduction/development toxicity potential of the test substance in rats according to OECD Guideline 422 and 421, OPPTS 870.3650 and 870.3550, in compliance with GLP. The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 100, 300 and 1000 mg/kg bw. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females that delivered were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 5-7 days of lactation (for 41-47 days). Females that failed to deliver healthy offspring were exposed for 41 days. Formulations were analyzed once during the study to assess accuracy and homogeneity and stability. The following observations and examinations were evaluated: mortality / viability, clinical signs, functional observations and locomotor activit, body weight and food consumption, clinical pathology, macroscopy at termination, organ weights and histopathology on a selection of tissues. Macroscopic and microscopic evaluations in females included ovaries, uterus and vagina. Ovaries and uterus (including cervix) were weighted. The number of former implantation sites and corpora lutea were recorded for all paired females. Macroscopic and microscopic examinations in males included prostate gland, testes, seminal vesicles and epididymides. The prostate gland, testes, seminal vesicles including coagulating glands and epididymides were weighted. Additional slides of the testes were realised to examine the staging of spermatogenesis. The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups were analyzed as well. Mating index (%), fertility index (%), conception index (%), gestation index (%) and duration of gestation as well as general reproduction data including male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Cage debris of pregnant females were examined for evidence of premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were reported as well. Mortality/viability, clinical signs, body weight and sex ratio as well as external and internal macroscopic examination of the pups were performed. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature protected from light. No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw). No treatment-related toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, number of implantation sites and corpora lutea, spermatogenic profiling, and histopathological examination of reproductive organs). Regarding the developmental results; at 1000 mg/kg bw, there was a trend in decreased pup weights noted, however the change was not statistical significant and the mean values were decreased by 6 % and 8 % on Days 1 and 4, respectively when compared to concurrent controls. Therefore, the lower pup weight was not considered to be adverse. No treatment-related toxicologically significant changes were noted in any other developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy). For details on other systemic effects refer to the discussion under section 5.6.1.1 of the CSR. Under the study conditions, the NOAEL for reproduction and developmental toxicity in rats were determined to be at least 1000 mg/kg bw (de Raaf-Beekhuijzen, 2016).