AERO 5100 PROMOTER

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (OECD 422), rat: NOAEL fertility: 15 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Dec 2016 - 19 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 (males) and 13 (females) weeks
- Housing: Animals were housed in groups of five in Makrolon type-4 cages (height 18 cm) for pre-test and pre-mating period and for recovery animals throughout the complete study period. During the mating period, females were housed with sexually mature males (1:1) in Macrolon type-3 cages. Post-mating males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust was used as bedding with paper enrichment.

- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD, WATER AND BEDDING QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item and the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 post-coitum
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions 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%.

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

Duration of treatment / exposure:

Males of main groups and recovery groups were treated for 32 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females of main group that delivered were treated for 52-66 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 41-45 days. Females of recovery groups were exposed during the same period as females of main group, until at least the first scheduled necropsy of main group high dose females (i.e. 41 days). Routinely, females that are littering are left undisturbed. In this study, female nos. 51, 52, 59 (control group), 66, 67, 71 (low-dose group), 80, 83 and 85 (mid-dose group) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Frequency of treatment:

once daily, 7 days/week, approx. the same time each day with a max. of 6 h difference between the earliest and latest dose

Details on study schedule:

not applicable for an OECD 422 study

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 (main groups)
5 (recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previously conducted 28-day repeated dose toxicity study dose levels of 15, 50 and 150 mg/kg bw /day were tested and a No Observed Adverse Effect level could not be determined. In this study decreased thymus weights (dose related in males) and slight (15-50 mg/kg bw/day) to moderate (150 mg/kg bw/day) thymus involution/atrophy were observed. At 50 mg/kg bw/day increased liver weights were observed in both sexes. In the 150 mg/kg bw/day group several signs of systemic toxicity were noted. Therefore, based on results of 28-day repeated dose toxicity study, in present study dose levels of 3, 15 and 75 mg/kg bw/day were selected.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality/viability
- Cage side observations included: All animals were observed for general condition and clinical signs at least once daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals during treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weight on Day 1 (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

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

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous for all females of main group.

Sperm parameters (parental animals):

Parameters examined in male parental generations (P0):
testis weight, epididymides weight, seminal vesicle weight, histopathological examination of testes to examine staging of spermatogenesis (control and high dose group only and main group males that failed to sire)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; 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.

OTHER:
THYROID HORMONE ANALYSIS:
- Time schedule for collection of blood: Blood samples were collected on the day of necropsy (PND 13-15)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 2 pups per litter
- Parameters examined: thyroxine (T4)

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
All males of the main group were sacrificed after mating (min. of 28 days of dose administration) and females of the main group were sacrificed on Day 14-16 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Animals were sacrificed in extremis if pain, distress or discomfort was considered not transient in nature or was likely to become more severe. 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.

ORGAN WEIGHTS: Yes
Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. 5 males and 6 females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed for selected animals: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles (including coagulating glands), spleen, testes, thymus, thyroid (including parathyroid if detectable), uterus (including cervix). For all remaining animals: epididymides, prostate, seminal vesicle (including coagulating glands), testes, thyroid (including parathyroid if detectable)

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation:
Samples of the following tissues and organs were collected for selected 5 animals per group and sex of recovery and main groups and were fixed in 10% buffered formalin or modified Davidson's solution (epididymides and testes): adrenal glands, brain (cerebellum, mid-brain and cortex), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, esophagus, eyes (with optic nerve and Harderian gland), heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodes (mandibular, mesenteric), ovaries, Peyer´s patches, pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, mid-thoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid (including parathyroid), trachea, urinary bladder, uterus, vagina, all gross lesions

5 males and 5 females were randomly selected from the main vehicle control and high dose group in addition to all recovery animals for tissue preparation and examination by an pathologist. Additionally, the follwing organs/tissues were examined:
- slides of testes of the selected 5 main group males of vehicle control and high dose groups and all main group males that failed to sire to examine staging of spermatogenesis
- preserved organs of animals euthanized in extremis and of all males of main group that failed to sire to examine staging of spermatogenesis
- all gross lesions of all dose groups
- thymus and spleen of all selected 5 main group animals of low- and mid-dose group and all recovery animals (males and females) and liver and mesenteric lymph nodes of all selected 5 main group animals of low- and mid-dose group and all recovery animals (males), based on (possible) treatment-related changes in these organs in high dose group
- the reproductive organs of all main group males that failed to sire and all main group females that failed to deliver healthy pups

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed

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 (many-to-one t-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 (many-to-one rank 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) in the summary tables. 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 (%) = (number of females mated / number of females paired) x 100
Fertility index (%) = (number of pregnant females / number of females mated) x 100
Gestation index (%) = (number of females bearing live pups / pregnant females) x 100

Offspring viability indices:

Post-implantation suvival index (%) = number of offsping born / number of uterine implantation sites
(Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.)
Live birth index (%) = (live offspring on Day 1 after littering / number of offspring born) x 100
Percentage live males at first litter check (%) = number of live males / number of live pups
Percentage live females at first litter check (%) = number of live males / number of live pups
Viability index (%) = (live offsping on Day 4 (before culling) / number of offspring on Day 1 after littering) x 100
Lactation index (%) = number of live offspring on Day 13 after littering / number of live offspring on Day 4 (after culling)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 75 mg/kg bw/day, rales were observed in 5 out of 15 males, ranging from one day to 20 days during the treatment period.
Salivation seen after dosing among animals of the 3 (one male for one day only), 15 and 75 mg/kg bw/day dose groups during the treatment period 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).
Incidental findings that were noted included focal erythema, scales, scabs and alopecia. Rales were noted for individual males in the vehicle control and 15 mg/kg bw/day dose groups for one day only. 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.
No findings were noted during detailed clinical observations in this study.

Dermal irritation (if dermal study):

not examined

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 item. One female at 3 mg/kg bw/day was sacrificed due to delivery difficulties on Day 23 post-coitum.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse changes in body weights and body weight gain were noted up to 75 mg/kg bw/day.
Treatment related reduced body weight gain was observed at 75 mg/kg bw/day in males from the first week of treatment onwards and in females starting in the second week of treatment. Body weights of 75 mg/kg bw/day treated males were approximately 6% and 8% lower compared with concurrent controls at the end of treatment and end of recovery, respectively. In 75 mg/kg bw/day treated recovery females body weights were approximately 8% lower compared with concurrent vehicle controls at the end of the mating period (partially due to body weight gain of one female which mating was overlooked). At the end of recovery, the difference was approximately 2%.
During the post-coitum phase, females treated at 75 mg/kg bw/day had lower body weights compared to the control group starting from Day 0 post coitum (statistically significant from Day 4 post-coitum onwards). Females in this dose group had similar body weight gain as concurrent controls up to Day 11 post-coitum, which subsequently stagnated from Day 14 post-coitum onwards. The lower body weights from Day 0 post-coitum onwards were resulting from the reduced body weight gain during the first weeks of treatment, whereas the reduced body weight gain during the last part of the post-coitum phase was considered to be due to the pregnancy status of these females (i.e. implantation sites only).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effect on food consumption was observed up to 75 mg/kg bw/day.
Food consumption was reduced in the first week of treatment at 75 mg/kg bw/day compared with the concurrent controls (not statistically significant).
During the post-coitum phase food consumption before or after allowance for body weight was slightly reduced in 75 mg/kg bw/day treated females compared to the concurrent controls. This was considered not adverse but due to the pregnancy status of these females (i.e. implantation sites only). Values at 75 mg/kg bw/day were in line with that of untreated, nulliparous females of this strain and age.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Note: Females at 75 mg/kg bw/day had no live offspring and the recovery females were not mated. Several haematology parameters are known to be influenced by physiological status (lactating versus no former pregnancy or former pregnancy but no offspring). Therefore, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females.
The following (statistically significant) changes in haematology parameters distinguished treated animals from control animals at the end of treatment:
- (Slightly) lower percentage of reticulocytes at 75 mg/kg bw/day at the end of treatment (statistically significant in males only) and at the end of recovery (statistically significant in females only)
- Lower mean corpuscular volume (MCV) in 75 mg/kg bw/day treated males (end of treatment) and lower mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females at the end of treatment and after recovery
The above changes remained within the normal range for rats of this age, strain and applicable physiologically status and were considered not adverse.
Values for the percentages of neutrophils and lymphocytes in 3 and 15 mg/kg bw/day treated females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of a treatment-related distribution not considered to be toxicologically relevant.
The statistically significantly decrease in red blood cells in 15 mg/kg bw/day treated females was within the normal limits and at 75 mg/kg bw/day red blood cell counts appeared unaffected by treatment, therefore the decrease at 15 mg/kg bw/day was not attributed to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Note: Females at 75 mg/kg bw/day had no live offspring and the recovery females were not mated. Several clinical biochemistry parameters are known to be influenced by physiological status (lactating versus no former pregnancy or former pregnancy but no offspring). Therefore, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females at the end of treatment.
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Increased total bilirubin levels at 75 mg/kg bw/day in males
- Increased levels of total protein and albumin in 75 mg/kg bw/day treated females
These statistically significant changes at 75 mg/kg bw/day were not considered to be toxicologically significant as they remained within the range considered normal for rats of this age and strain.
Values at the end of recovery achieving a level of statistical significance when compared to controls, were considered of no toxicological relevance in the absence of a treatment related effect at the end of treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of treatment, a potential treatment related decrease of the hind leg grip strength was observed in the 75 mg/kg bw/day treated recovery females, consequently grip strength assessment was repeated at the end of recovery for these animals. However, as this decrease was not observed in (combination with) the main 75 mg/kg bw/day treated females and the effect was not present at the end of recovery, this was considered to be a chance finding and not toxicologically relevant.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected main and recovery animals.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period a decreasing trend in activity over the duration of the test period.
The slight increase of ambulations in the 75 mg/kg bw/day treated males was not statistically significant and remained within the normal variation.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with the test substance were noted in the thymus of both sexes at 75 mg/kg bw/day and in the liver and spleen of males at 75 mg/kg bw/day.
Thymus: An increased incidence and/or severity of lymphoid atrophy was present in the thymus of males and females at 75 mg/kg bw/day. After a 14-day recovery period this finding showed complete recovery.
Spleen: A minor increase in incidence and severity of pigmentation, yellow-brown was recorded in males at 75 mg/kg bw/day. After a 14-day recovery period this finding showed complete recovery.
Liver: An increased incidence and/or severity of inflammatory cell infiltrate (slight), macrovesicular vacuolation (minimal) and hepatocellular hypertrophy (minimal) was recorded in the liver of males at 75 mg/kg bw/day. After a 14-day recovery period these findings showed complete recovery.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONE ANALYSES:
Note: Females at 75 mg/kg had no live offspring and the recovery females were not mated. To account for possible differences in thyroid hormone levels due to the physiological status of these females, results for non-lactating 75 mg/kg bw/day treated females were compared to non-mated recovery females at the end of treatment.
Serum levels of T4 in males were statistically significantly decreased at 75 mg/kg bw/day at the end of treatment. At the end of recovery T4 serum levels remained reduced compared with concurrent controls (not statistically significant). At the end of treatment serum levels of TSH were unaffected in 75 mg/kg bw/day treated males, therefore the significantly reduced serum levels at the end of recovery in males of this dose group were considered not toxicologically relevant.
The T4 serum levels in females were unaffected by treatment at 75 mg/kg bw/day. At the end of treatment, serum TSH levels of 75 mg/kg bw/day treated females were slightly increased compared to the non-mated recovery females of the control group. As this effect was only slight and not statistically significant, this was considered not treatment related. The lower TSH serum levels for both controls and 75 mg/kg bw/day treated recovery females at the end of recovery were considered to be a chance finding.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Adverse treatment related effects on the length and regularity of the estrous cycle were noted in 75 mg/kg bw/day treated females. At this dose level, three main group females had an irregular cycle during the treatment phase. One recovery female also had an irregular cycle during the treatment phase, and additional female was found acyclic.
At 3 and 15 mg/kg bw/day, respectively one and two females had an irregular cycle during the premating phase. These incidences remained within the normal range for this strain and age and was therefore considered not to be adverse. Moreover, these females had normal sized litters with live pups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was considered not to be affected by treatment. All females showed evidence of mating.
Precoital time was not considered to be affected by treatment.
Fertility index was not considered to be affected by treatment up to 75 mg/kg bw/day. All mated females were pregnant, except for one female, treated at 15 mg/kg bw/day. 1 out of 10 couples of the 3 mg/kg bw/day group and all couples of the 75 mg/kg bw/day group had no live offspring. All these females showed remnants of former implantation sites. No abnormalities were seen in the reproductive organs of males and females, which could account for their lack of offspring.
Gestation index and duration:
All females treated at 75 mg/kg bw/day had implantation sites only. Gestation index and duration of gestation were not considered to be affected by treatment up to 15 mg/kg bw/day.
Number of implantation sites was not considered to be affected by treatment up to 75 mg/kg bw/day.
Litter size: Litter size was not considered affected by treatment up to 15 mg/kg bw/day. The litter size in the control group was relatively low, this was considered a chance finding.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects observed at 75 mg/kg bw/day (highest dose tested)

Dose descriptor:

LOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproduction observed at 15 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

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.
For the pup frome a control female that was missing on Day 2 of lactation, less milk in the stomach was noted at first litter check. And for the pup of one female treated at 3 mg/kg bw/day, lean and cold appearance, absence of milk and wound/scabs on the right hind leg were observed prior to the moment it went missing on Day 4 of lactation.
The nature and incidence of these and other 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):

As there were no females with live offspring in the high dose group, the developmental results described below relate to the groups treated up to 15 mg/kg bw/day.

Post-implantation suvival index: The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment up to 15 mg/kg bw/day. For one female (3 mg/kg bw/day), the number of pups was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 16 of lactation. No toxicological relevance was attached to this finding in the current study.

Live birth index: 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. Two control female had respectively 1 and 6 dead pups at first litter check. This was considered to be a chance finding.

Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment.
Two pups of the control group and one pup at 3 and 15 mg/kg bw/day went missing on Day 2 or 4 of lactation. These pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. One pup at 3 mg/kg bw/day was euthanized at Day 4 of lactation as it was in poor condition (lean, less milk, cold and pale).

Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not considered to be affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment up to 15 mg/kg bw/day.

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

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment up to 15 mg/kg bw/day. Treatment up to 15 mg/kg bw/day had no effect on areola/nipple retention, nipples were not observed at PND 13 for any of the examined male pups.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups at 3 and 15 mg/kg bw/day were not considered to be affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Sex ratio was not considered to be affected by treatment.
Parturition/maternal care: No treatment related effects on parturition or maternal care were observed up to 75 mg/kg bw/day.
One female at 3 mg/kg bw/day was sacrificed due to delivery difficulties on Day 23 post-coitum. At necropsy, 10 dead fetuses were found for which no abnormalities were observed. This was considered incidental and not related to treatment.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

LOAEL

Remarks:

development/teratogenicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Dose descriptor:

NOAEL

Remarks:

development/teratogenicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on development observed

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: not specified

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was ≥ 75 mg/kg bw/day because no adverse systemic effects were observed up to 75 mg(kg bw/day the highest dose tested.
The NOAEL for reproduction was derived to be 15 mg/kg bw/day based on increased incidence of females with an irregular cycle in combination with an acyclic female at 75 mg/kg bw/day.
The NOAEL for development was derived to be 15 mg/kg bw/day based on complete post-implantational loss at 75 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity on reproduction

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (Hartman-Van Dycke, 2017). Based on a previous 28-day repeated dose toxicity study, groups of 10 male and 10 female Han Wistar rats were treated once daily by gavage with the test substance at 3, 15 and 75 mg/kg bw/day or the vehicle (corn oil) only. For recovery,5 additional animals/sex were added to the each of the control and high dose group.Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 32 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 52-66 days). Females that failed to deliver healthy offspring were treated for 41-45 days. The recovery males and females were not subjected to mating and were allowed a 2-week treatment-free period. The recovery females were treated for 41 days before start of recovery. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment; grip strength females repeated at the end of recovery), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy (Main females only)), clinical pathology (end of treatment and end of recovery), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction parameters were determined: mating, fertility, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care.

No adverse parental toxicity was observed up to the highest dose level tested (75 mg/kg). Treatment related effects on the length and regularity of the estrous cycle were noted in 75 mg/kg bw/day treated females. The increased incidence of females with an irregular cycle in combination with the occurrence of an acyclic female in the high dose group was considered as an adverse effect of treatment. All other parameters regarding fertility toxicity were without abnormal findings.

No reproduction toxicity was observed up to 15 mg/kg bw/day.

In conclusion,based on cycle irregularities and acyclicity in female rats at 75 mg/kg bw/day group, a NOAEL (fertility) of 15 mg/kg bw/day for reproductive toxicity was derived in this study. A NOAEL (fertility) of ≥ 75 mg/kg bw/day was derived for males since no adverse effects were observed up to the highest dose tested.

 

Effects on developmental toxicity

Description of key information

Oral (OECD 422), rat: NOAEL development: 15 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity on development

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (Hartman-Van Dycke, 2017). Based on a previous 28-day repeated dose toxicity study, groups of 10 male and 10 female Han Wistar rats were treated once daily by gavage with the test substance at 3, 15 and 75 mg/kg bw/day or the vehicle (corn oil) only. For recovery,5 additional animals/sex were added to the each of the control and high dose group.Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 32 days). The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 52-66 days). Females that failed to deliver healthy offspring were treated for 41-45 days. The recovery males and females were not subjected to mating and were allowed a 2-week treatment-free period. The recovery females were treated for 41 days before start of recovery. Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 5 hours at room temperature.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment; grip strength females repeated at the end of recovery), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy (Main females only)), clinical pathology (end of treatment and end of recovery), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following developmental parameters on the offsprings were determined: sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

No adverse parental toxicity was observed up to the highest dose level tested (75 mg/kg).

None of the high dose females (75 mg/kg bw/day) had live offspring. All females of this group showed remnants of former implantation sites which indicate an embryotoxicity. This is supported by the fact that no abnormalities were seen in the reproductive organs of males and females which could account for this adverse effects on the early development of the offspring. All other examined parameters on the offspring showed no abnormal findings.

No developmental toxicity was observed up to 15 mg/kg bw/day.

In conclusion,based on absence of live pups born at 75 mg/kg bw/day group, a NOAEL of 15 mg/kg bw/day for developmental toxicity/teratogenicity was derived in this study.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance meet the criteria for classification as Repr. 2 (H361d) according to Regulation (EC) No 1272/2008 due to total post-implantational loss at 75 mg/kg bw/day. A classification for fertility (H360f) does not seem warranted because the effects on cycle irregularities and acyclicity in female rats showed no negative outcome as all females in the high dose group had implantation sites.