Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Aug - 22 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-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:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), 2000
Deviations:
no
GLP compliance:
yes
Remarks:
LAUS GmbH: Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 316-356 g (males), 205-230 g (females)
- Housing: Pre-mating: Groups of 5 animals per sex per cage in Makrolon plastic cages (MIV type, height 18 cm); Mating: 1:1 in
Macrolon plastic cages (MIII type, height 18 cm); Post-mating: Groups of 5 males per cage in Macrolon plastic cages (MIV type, height 18 cm), females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Sterilized sawdust was used as bedding material, paper as cage-enrichment/nesting material. During locomotor activity monitoring, animals were housed individually in a Hitemp polycarbonate cage (48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted rodent diet SM R/M-Z (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
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 vehicle.

VEHICLE
- Justification for use and choice of vehicle: based on trial formulations performed at testing laboratory
- Concentration in vehicle: 1, 3 and 10 % (w/v)
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: intravaginal copulatory plug or sperm in vaginal lavage referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in plastic Makrolon cages (MIII type, height 18 cm) with paper as nesting material
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 85-110% 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%.
The concentration in formulations was in the acceptable range (100 ± 15% of nominal concentration) for all samples. Stability was shown for 5 h at room temperature at nominal concentrations 10 g/L and 100 g/L. Accurate preparation of the suspensions, homogeneous distribution of the test item in the suspensions and stability over 5 h at room temperature were demonstrated by analysis. The test item was separated with a Phenomenex Gemini-NX 2x100 mm column and detected with tandem mass spectrometry.
Duration of treatment / exposure:
males: for 29 days, starting 2 weeks before mating, during mating and up to the day prior scheduled necropsy
females: for 43-45 days, during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy)
Frequency of treatment:
once daily, 7 days/week
Details on study schedule:
not applicable for an OECD 422 study
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study, in which animals were orally exposed to 500 and 1000 mg/kg bw/day for 10 days (2014). No mortality, macroscopic findings and no effects on organ weights (liver and kidney) were observed. Uncoordinated movements on Day 4 at 500 and 1000 mg/kg bw/day and white discoloration of feces on Day 6 at 1000 mg/kg bw/day were noted. Slightly lower mean body weight gain and lower mean body weights were recorded at the highest dose level. Furthermore, lower mean absolute food consumption was observed at 500 and 1000 mg/kg bw/day. Therefore, 100, 300 and 1000 mg/kg bw were selected as the dose levels for the main study. The peak effect of occurrence of clinical signs occurred at 1 to 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests are to be conducted after dosing in the main study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, between 1 and 3 hours 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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption was determined 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 on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: 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 (for details refer to IUCLID section 7.5.1)
Sperm parameters (parental animals):
Samples of testes from 5 males of control and high dose group were stained with PAS/hematoxylin to assess staging of spermatogenesis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, postnatal mortality, presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD PUPS:
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol. All pups were sexed and descriptions of all external 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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals: All animals which delivered on lactation Days 5-7. Females with total litter loss within 24 h of litter loss.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues and organs were collected and fixed in 10% buffered formalin from selected 5 animals/sex/group: adrenal glands, (aorta), brain - cerebellum, mid-brain, cortex, caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve (if detectable) and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, (lacrimal gland, exorbital), (larynx), liver, lung, infused with formalin, lymph nodes - mandibular, mesenteric, (nasopharynx), (esophagus), ovaries, (pancreas), Peyer's patches [jejunum, ileum] if detectable, pituitary gland, preputial gland, prostate gland, rectum, (salivary glands - mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, (skin), spinal cord -cervical, midthoracic, lumbar, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, (tongue), trachea, urinary bladder, uterus, vagina and all gross lesions (tissues/organs mentioned in parentheses were not examined, since no signs were noted at macroscopic examination)

The following tissues and organs were collected and fixed in 10% buffered formalin from all remaining animals and female with total litter loss: cervix, clitoral gland, coagulation gland, epididymides, female mammary gland area, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, vagina and all gross lesions

Organ weight was determined from the following tissues/organs from selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, liver, kidneys, ovaries, spleen, testes, thymus, seminal vesicles including coagulating glands, thyroids including parathyroids, prostate and uterus

Organ weight was determined from the following tissues/organs from all remaining males: epididymides, testes

The following organ and tissue samples were processed, embedded and cut at a thickness of 2-4 µm. Slides were stained with haematoxylin and eosin and examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of the control and high dose group were examined.
- The additional slides of the testes of the selected 5 males of the control and high dose group to examine staging of
spermatogenesis (stained with PAS/haematoxylin).
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, ovaries, uterus, and vagina) of female 42 that had a total litter loss.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external 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.
Reproductive indices:
Mating index (%) = (number of females mated / number of females paired) x 100
Fertility index (%) = (number of pregnant females / number of females paired) x 100
Conception index (%) = (number of pregnant females / number of females mated) x 100
Gestation index (%) = (number of females bearing live pups / number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at first litter check = (number of live male pups at first litter check / number of live pups at first litter check) x 100
Percentage live females at first litter check = (number of live female pups at first litter check / number of live pups at first litter check) x 100
Percentage of postnatal loss = (number of dead pups before planned necropsy / number of live pups at first litter check) x 100
Viablity index (%) = (number of live pups before planned necropsy / number of pups born alive) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100, 300 and 1000 mg/kg bw/day, both sexes: Increase incidence of uncoordinated movements, erythema of the ears and pale feces. The signs were not considered to be adverse.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

A dose-dependent increase in the incidence of uncoordinated movements, erythema of the ears and pale feces was noted. The incidence of uncoordinated movements was higher for animals in all treated groups, though they were also observed occasionally in the control group. Uncoordinated movements were noted for all animals at 300 and 1000 mg/kg bw/day, for 5 males and 9 females at 100 mg/kg bw/day and for 4 control males and 1 control female. This sign was not considered to be adverse as there was no toxicologically relevant effects seen for grip strength or locomotor activity.

Erythema of the ears was seen for 9 males and 7 females at 1000 mg/kg bw/day, 2 females each in the 100 and 300 mg/kg bw/day groups and 1 control female. This was clearly treatment-related, but in the absence of signs of ill health, it was not considered to be adverse.

Pale feces were noted for most or all animals at 300 and 1000 mg/kg bw/day. Pale feces were also noted for a few females at 100 mg/kg bw/day, though the incidence was much lower. This was likely related to the color of the cloudy, white formulations and was not adverse.

Scabbing or alopecia, rales, salivation, pale appearance (over 2 days for 1 male at 300 mg/kg bw/day), red, orange or brown staining of the neck and/or vagina, chromodacryorrhoea, piloerection (seen for a single female at 1000 mg/kg bw/day) were incidental findings seen for control and/or treated animals. 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 toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION
Absolute food consumption was higher during the lactation period for animals in all treated groups (significantly higher for females at 100 and 1000 mg/kg bw/day). This was secondary to relatively low food consumption for control females. As such, these differences were not considered attributable to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

The spermatogenic staging profiles were normal for all males examined.

The gestation index and duration of gestation were similar between control and treated animals. All females were pregnant and delivered live pups.

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.

ORGAN WEIGHTS
No toxicologically relevant changes in organ weights and organ to body weight ratios were seen. Absolute brain weights were significantly lower for females at 1000 mg/kg bw/day. No toxicological relevance was attributed to this, as the difference from controls was slight, no differences in brain to body ratios were seen and no corresponding microscopic findings were seen.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were attributable to treatment. Incidental findings noted for control and/or treated animals included nodule of the epididymis, reddish or dark red foci on or discoloration of the thymus, reduced size of the thymus, pelvic dilation of the kidneys, black discoloration and reduced size of the liver papillary process, thickened duodenum wall, tan focus on the clitoral gland, watery clear cysts on the kidney, enlarged mandibular lymph node, and alopecia. These were 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. As such these were not considered treatment related.

HISTOPATHOLOGY
There were no test item-related microscopic findings. The histologic changes recorded were considered to be incidental findings. There was no test item-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations.

There was one female of the control group with a total litter loss. A cause for the loss of this litter could not be established from the sections examined.

OTHER:
For results on heamatology, clinical chemistry, neurobehavioural examination refer to IUCLID section 7.5.1.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse systemic effects and no adverse effects on reproduction up to and including the highest dose tested
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
only external
Histopathological findings:
not examined
VIABILITY (OFFSPRING) / EARLY POSTNATAL DEVELOPMENT
There were no adverse effects on early postnatal pup development (including viability index and sex ratio) with treatment up to 1000 mg/kg bw/day and clinical signs, body weight and external macroscopic examination did not reveal treatment-related findings. Mean litter sizes (living pups) were 7.6, 10.6, 10.5 and 10.3 pups per litter in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. The mean litter size for control females was lower than the historical control mean of 11.7 pups.

No treatment related effects on pup mortality up to 1000 mg/kg bw/day was observed. In the control group were 6 dead pups at first litter check and 10 pups lost during the first days of lactation. Three of these dead pups were from one female who lost her entire litter by Day 3. There were no dead pups at the first litter check for the remaining groups. Postnatal loss during the first days of lactation consisted of 8, 2 and 1 dead pups for the 100, 300 and 1000 mg/kg bw/day groups, respectively.

The incidence of dead pups at first litter check was much higher for control animals than treated ones. Similarly, the postnatal loss was significantly lower and the viability index was significantly higher for females at 300 and 1000 mg/kg bw/day. These were not considered to be toxicologically relevant as the number of deaths were lower in the 300 and 1000 mg/kg bw/day groups. The number of deaths in the control and 100 mg/kg bw groups were unusually high compared to historical control data. The treated groups were also compared against a robust historical control database thus all data could be thoroughly evaluated.

CLINICAL SIGNS
Clinical signs noted for pups that died or went missing included a wound on the back or belly, autolysis and cannibalism. Clinical signs for pups surviving until the scheduled necropsy were incidental in nature and included scab on the head, blue spot on the flank(s) and endorotation of the left hindleg. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not toxicologically relevant.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic findings noted for pups that died or went missing included autolysis and cannibalism. Macroscopic findings noted for surviving pups included scab on the head, blue spot both flanks, ink in the abdominal cavity and endorotation of the left hindleg. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development of offspring up to and including the highest dose tested
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

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 (Zmarowski, 2015). Ten Sprague Dawley rats per sex and dose were treated via gavage with 100, 300, and 1000 mg/kg bw/day, respectively. The control group received the vehicle propylene glycol. Male animals were treated for 29 days, starting 2 weeks before the mating period, during mating and up to the day prior scheduled necropsy. Females were treated for 43-45 days, i.e. during 2 weeks before mating and during the mating period until successful copulation, and during gestation through at least day 4 after parturition. The doses were selected on the basis of data from a range finding study.

In the main study, no mortality occurred during the study period that was considered to be related to treatment with the test substance. Animals in all treated groups, but particularly at 300 and 1000 mg/kg bw/day had higher incidences of clinical signs including uncoordinated movements, erythema of the ears and pale feces. As these signs were also observed for control animals (uncoordinated movements and erythema), reflected the color of the test substance formulations (pale feces) and occurred in the absence of any signs indicative of treatment related toxicity, they were considered treatment related but not adverse. No treatment-related effects were noted in functional observations, body weight, food consumption, haematology, biochemistry, or necropsy in males or females in any test substance group.

Therefore the NOAEL for parental systemic toxicity was ≥ 1000 mg/kg bw/day.

In parental animals, no effects on reproductive function (qualitative sperm staging) or performance (male and female mating, fertility, conception indices, precoital time, and number of corpora lutea and implantation sites, gestation index and duration, parturition and maternal care) were observed, compared with the control animals. The testis weight, epididymis weight, and histological examination of the testes and epididymis in males as well as the weight and histological examination of the uterus and ovaries in females did not reveal any substance-related effects in the parental animals.

Therefore, a NOAEL for parental fertility of ≥ 1000 mg/kg bw/day was derived for male and female rats.


Short description of key information:
Oral (OECD 422), rat: NOAEL reproduction ≥ 1000 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
The reliable GLP compliant OECD Guideline study was chosen.

Effects on developmental toxicity

Description of key information
Oral (OECD 422), rat: NOAEL reproduction ≥ 1000 mg/kg bw/day
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 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

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 (Zmarowski, 2015). Ten Sprague Dawley rats per sex and dose were treated via gavage with 100, 300, and 1000 mg/kg bw/day, respectively. The control group received the vehicle propylene glycol. Male animals were treated for 29 days, starting 2 weeks before the mating period, during mating and up to the day prior scheduled necropsy. Females were treated for 43-45 days, i.e. during 2 weeks before mating and during the mating period until successful copulation, and during gestation through at least day 4 after parturition.

There were no adverse effects on early postnatal pup development (including the number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio) with treatment up to 1000 mg/kg bw/day and clinical signs, body weight and external macroscopic examination did not reveal treatment-related findings.

Thus the NOAEL for developmental toxicity/teratogenicity is considered to be ≥ 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
The reliable GLP compliant OECD Guideline study was chosen.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification. No adverse effects were noted up to the limit dose in an OECD 422 guideline study.

Additional information