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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Oct - 06 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Comission Regulation (EC) No. 440/2008, L142, Appendix Part B
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: males / females: 10-11 weeks
- Weight at study initiation: males mean: 271 g, females mean: 194 g
- Fasting period before study: no data
- Housing: the animals were kept individually in IVC cages (except during the mating period), type III H, polysulphone cages on Altromin saw fibre bedding; females were separated and housed individually after confirmation of the mating
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0856), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- 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: The test item formulation was prepared freshly on each day of administration. After cooling the test item in a refrigerator for 10 minutes at 2-8 ºC, the test item was grinded to a fine powder using a mortar and pestle or laboratory electric mill. When mortar and pestle was used for grinding, it was ensured that not too much force was applied during the grinding and grinded with little force in circulating movements to prepare a very fine powder. The powdered test item was weighed into a tared plastic vial or container on a suitable precision balance and then the vehicle (corn oil) was added to give the appropriate final concentration of the test item. The formulation vials/container was shaken by hand for short period to ensure proper homogenistation of the formulation.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle has been selected on the basis of the test item’s characteristics and the results of the dose range finding study.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Batch no. (if required): MKBH 4894 V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- In case of unsuccessful mating, re-mating of females with proven males of the same group was considered.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, stability and homogeneity of the formulated samples were analytically verified using HPLC with UV detection.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) (Total 16 samples) for all dose groups. The mean recoveries observed in the low mid and high dose groups were 98.7 %, 103.8 % and 98.0 % of the nominal concentration, respectively.
Stability of formulation samples was investigated in week 1 for low and high dose groups. Two aliquots per dose were analyzed immediately after preparation, another two per dose were stored for 6 hours at room temperature and analyzed thereafter. After 6 hours storage at room temperature recovery compared to starting value was 105.0 % and 134.9 % for low and high dose samples. Since the initial consistency of freshly prepared formulation samples is a suspension transforming within approx. an hour into a jellylike lump it is assumed that paralelly the density of the formulation, and therefore its concentration increases. This effect is observed at the higher concentration only. Because animals in the main study were administred within 1 hour, the 6 hour stability measurement was not relevant for the study and therefore the stability of samples at the concentration of high dose group was investigated again for the storage period of 1 hour. Investigation of 1 hour stability of high dose samples confirmed that the samples are stable and revealed 99.3 % recovery.
Samples for homogeneity analysis were taken in week 1 and week 5 of the study from top, middle and bottom of low, mid and high dose preparations. The mean recovery observed for low dose group was 97.2 and 94.1 % of the nominal value, for mid dose group was 101.0 and 106.3 % and 100.4 and 100.1 % for high dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 6.4 and 5.7 % in low dose group, 8.8 and 0.6 % in mid dose group and 2.0 and 0.7 % in high dose group.
Duration of treatment / exposure:
females: 54 days (14 days of pre-mating and maximum 14 days of mating, during the gestation period and up to post-natal day 3)
males: 28 days
Frequency of treatment:
daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300, 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: The choice of test substance concentrations was based on a dose range finding study (BSL project no. 123581). The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, on the corresponding days of the body weight measurements, except during the mating period in female animals and the mating and post-mating period in male animals.
- Mean weekly diet consumption per sex calculated as g food/kg body weight/week: Yes
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Parameters examined in P male parental generation:
- weights of testis, epididymis, prostate, seminal vesicles and coagulating glands
- for the testes, a detailed qualitative examination was made taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in the offspring:
sex of pups, body weights, stillbirths, live births, runts and the presence of gross abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were were sacrificed after 28 days of dosing.
- Maternal animals: All surviving animals were sacrificed on day 4 of lactation.

GROSS PATHOLOGY: Yes (All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.) The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

HISTOPATHOLOGY: Yes (on the following preserved organs and tissues of 5 randomly selected male and female animals of the control and high dose groups: adrenal glands, brain (cerebrum, cerebellum and pons), bone with bone marrow (sternum), epididymides, eye, gross lesions, heart, kidneys, liver, lungs and trachea, lymphnodes, mammary glands, oesophagus, ovaries, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, pituitary gland, prostate and seminal vesicles with coagulating glands as a whole, skin, small and large intestines (including Peyer´s patches), spinal cord, spleen, stomach, testes, thymus, thyroid, urinary bladder, uterus with cervix, vagina.
The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for eyes, testes and epididymides which were preserved in modified Davidson’s Solution.)
The wet weight of the following organs of 5 sacrificed males and 5 females randomly selected from each group was recorded as soon as possible (Paired organs were weighed separately. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded): adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate (seminal vesicles and coagulating glands), spleen, testes, thymus, thyroid/parathyroid glands, uterus with cervix
Postmortem examinations (offspring):
SACRIFICE
- The offspring was sacrificed at 4 days of age.
- These animals were carefully examined externally for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal cavities and their contents.
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.5.01 and V.6.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
- Corpora lutea, number of implantation sites, percentage of pre-implantation loss and post-implantation loss
- Copulation Index (%) = (No. of rats copulated / No.of pairs) X 100
- Fertility Index (%) = (No. of females pregant / No.of females copulated) X 100
- Delivery Index (%) = (No. of dams with live newborns / No.of pregnant dams) X 100
Offspring viability indices:
- Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100
Clinical signs:
no effects observed
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:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred in the control or any of the dose groups during the treatment period of this study.
No test item related clinical signs were observed in male and female animals during entire study period. Few predominant spontaneous clinical signs observed occasionally in male and female animals were alopecia on various body parts, red nasal discharge, slight to moderate salivation, abnormal breathing, slight piloerection and moving the bedding.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In both males and females, no statistically significant effect was observed on body weight and body weight gain in treatment groups during the most study phases. The only exception was a statistical significant decrease in male body weight gain in mid dose males during mating and postmating days 7-14. Due to the lack of dose dependency, this statistically significant decrease in male body weight gain was considered to be biologically irrelevant.
In both males and females, no statistically significant effect was observed on food consumption during the whole study period.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females in control and treatment groups showed evidence of copulation during 14 days mating period except one female from control group and 3 females from the mid dose group that did not show evidence of mating through vaginal smear. However, the animals were pregnant and littered normally. Successful mating resulted in 10 out of 10 pregnancies in the control, low, mid and high dose group.
No treatment-related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.
The group mean number of corpora lutea, number of implantation sites, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group. No effect on copulation index, fertility index and delivery index was observed in treatment groups when compared with controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males and females, no statistically significant difference in the absolute and relative organ weights was observed in the treatment groups except a statistically significant increase in relative liver weight in mid and high dose males and increase in relative adrenal weights in low and high dose males when compared with the control group. As the histopathological evaluation did not reveal any effect, the effects on liver and adrenal weights were considered to be spontaneous in nature and not treatment-related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy of males and females, macroscopic examination of the animals revealed few findings in males and females like small thymus (1/10 in low dose female), fluid filled uterus (1/10 in high dose female) and yellow spots on epididymis (2/10 in low dose and 2/10 in mid dose males). The yellow spots on epididymis were histologically confirmed to represent spermatic granuloma, a finding occasionally seen as spontaneous change in untreated rats of this strain and age.
Taken together, the gross pathological findings were spontaneous in nature and were assumed to be common background findings in this strain and as such not due to a systemic effect due to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The pathological evaluation did not reveal any test item-related effect on reproductive organs or on the other organs and tissues evaluated.
Spermatic granulomas were noted in 2/10 low dose and 2/10 mid dose males. Although this lesion was not noted in control males of this study, it was considered as a spontaneous change, as there was no dose relationship, and this finding is occasionally seen in untreated rats of this strain and age used at the test facility.
In the kidney, minimal numbers of debris-filled tubules were observed at the inner cortex of one single high dose male, but were not considered sufficient evidence of a test item related effect.
Minor inter-group differences were observed in the incidences of some findings. Minimal interstitial mononuclear cell infiltrates in the epididymis (2/10 in the control, 1/10 in the low dose, 5/10 in the mid dose and 7/10 in the high dose group), minimal cortical hyaline droplets in the kidney (2/10 in the control and 4/10 in the high dose group), minimally dilated glands in the trachea (1/10 in the control and 3/10 in the high dose group) and minimal or mild presence of germinal centers in the mesenteric lymph nodes (minimal: 1/10 in the control and 3/10 in the high dose group; mild: 2/10 in the control and 2/10 in the high dose group) were noted in males. In females, minimal basophilic tubules in the kidney (1/10 in the control and 3/10 in the high dose group) and minimal or mild sinusoidal erythrocytes in the mesenteric lymph nodes (minimal: 0/10 in the control and 3/10 in the high dose group; mild: 1/10 in the control and 0/10 in the high dose group) were observed. However these changes had a background incidence in the control group, the inter-group difference was not noted in both sexes and there was no tendency towards higher severity levels in the treated animals. Therefore, all of these inter-group differences were considered to be fortuitous in nature and not treatment-related.
Dose descriptor:
NOAEL
Remarks:
systemic, fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to 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
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
No pup mortality was observed in this study except one pup from the low dose group went missing on post-natal day 2 and was presumed to be cannibalized by the dam. Since this incidence of cannibalism was observed in one female and it was within the rat cannibalism rate, this incidence was not considered to be test item related.
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on post-natal day 0 and number of males, number of females, sex ratio and total number of live pups on post-natal day 4. However, statistically significant increase in number of males was observed in low dose group when compared with controls. Due to lack of dose dependency, this significant effect on group mean number of males was considered to be toxicologically irrelevant. No effect on viability index was observed in treatment groups when compared with controls.
There was increase in group mean number of still births observed in the high dose group when compared with control although statistical significance was not achieved. This increase in group mean number of still births was attributed to one female which gave birth to 11 dead pups. As this incidence just happened in one female and in other 9 females there were no incidences of still birth, this effect was considered to be incidental and not test item related.

CLINICAL SIGNS (OFFSPRING)
In the mid dose females, the hind legs of one pup were not moving on post-natal 0. This finding was considered to be spontaneous in nature and not related to the treatment with test item.

BODY WEIGHT (OFFSPRING)
Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on post-natal day 0 and 4 when compared with corresponding controls.

GROSS PATHOLOGY (OFFSPRING)
In the mid dose, one female pup was observed for dead tail tip and one pup was cold to touch and bluish in colour on post-natal day 4. These findings were considered to be spontaneous in nature and not related to the treatment with test item.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
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-IX, 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

A combined repeated dose/developmental toxicity screening study was conducted with UMA121 under GLP conditions according to OECD 422 in male and female Crl: WI(Han) rats (Takawale, 2013). Ten male and female rats each were once daily orally treated with test substance concentrations of 100, 300, 1000 mg/kg bw/day dissolved in corn oil. Females were treated 2 weeks before mating until day 3 of lactation (about 50 days) and the males for 28 days. Control animals were treated with the vehicle. No mortality and no test item related clinical signs were observed. Statistically significant effects noted were decreased body weight gain in mid dose males during mating and postmating days 7 -14 and increased relative liver weight in mid and high dose males as well as increased relative adrenal weights in low and high dose males. These effects were considered to be not biologically relevant due to the lack of dose dependency and as the histopathological evaluation did not reveal any effect on the respective organs. Gross pathology revealed few findings like small thymus in one low dose female and fluid filled uterus in one high dose female as well as yellow spots on epididymis in low and mid dose males. The yellow spots on epididymis were histologically confirmed to represent spermatic granuloma, a finding occasionally seen in untreated rats of this strain and age. As there was no dose-response relationship, spermatic granuloma were considered to be an incidental finding. Reproductive performance was assessed by determination of corpora lutea, number of implantation sites, percentage of pre-implantation loss and post-implantation loss, copulation index, fertility index and delivery index. None of these parameters were affected by test item treatment. Therefore, the NOAEL for fertility and developmental effects was determined to be ≥ 1000 mg/kg bw/day.


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

Justification for selection of Effect on fertility via oral route:
There is only one study available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
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

In accordance with Column 2 of Annex IX, Section 8.7 of Regulation (EC) No 1907/2006, a reproductive toxicity study is not required if the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure and there is no or no significant human exposure.

UMA121 is an insoluble and non-volatile resin with a molecular weight of > 538 g/mol. Despite manufacture and industrial formulation which take place in closed systems, UMA121, with a maximum concentration of 15% in the final product, is solely used by professionals resulting in the inclusion of the substance into a matrix. The test substance and its formulations are not used in spraying, drilling or grinding applications.

Based on the physico-chemical properties and the use pattern of UMA121, absorption via the oral, dermal or inhalation exposure route can be excluded (for details refer to section 7.1 of the technical dossier).

In a combined oral repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD 422 UMA121 was orally administered in doses of 100, 300 and 1000 mg/kg bw /day in corn oil by gavage to male and female rats (Takawale, 2013). To allow application, pre-cooled UMA121 had to be ground to a fine powder. No mortality and no test substance related clinical signs were observed up to and including the limit dose. In addition, gross pathology, organ weight determination and histopathology were without any test substance related findings. Developmental toxicity was assessed by determination of the total number of pups born, sex ratio, live pups, still birth and runt on post-natal day 0 and sex ratio and total number of live pups on post-natal day 4. None of these parameters was adversely affected in any of the treatment groups. In addition, litter weight determination and gross pathology of pups on post-natal day 4 were without any adverse findings.

Moreover, UMA121 was found to be not genotoxic in gene mutation tests in bacteria (Donath, 2012) and in mammalian cells (Zeller, 2013). No clastogenic effects were observed in an in vitro chromosome aberration test (Hofman-Hüther, 2013).

 

In summary, the available data on UMA121 (Takawale, 2013) did not indicate any evidence of test substance related effects on developmental toxicity up to and including the limit dose of 1000 mg/kg bw/day after the treatment of the pregnant females during gestation and lactation until post-natal day 4. In general, there was no evidence of local or systemic toxicity in any of the in vitro and in vivo tests available. Moreover, there is no evidence of absorption via the oral, dermal and inhalation route and human exposure is limited.

Thus, in accordance with Regulation (EC) No 1907/2006 Annex IX, Column 2, 8.7, a pre-natal developmental toxicity study does not need to be conducted. Furthermore, no additional study is justified for this endpoint based on animal welfare considerations according to Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.