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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility:

- 90-day oral repeated dose toxicity (OECD TG 408): No fertility effects in the male and female reproductive organs were observed at the maximum dose of 500 mg/kg bw

- 90 -day dermal /oral repeated dose toxicity studies in rat and mice effects cannot be used for deriving systemic and thus fertility effects.

- 28-day repeated dose (OECD TG 407): No fertility effects in the male and female reproductive organs were observed at the maximum dose of 1000 mg/kg bw

- An extended one-generation study is imposed in a final decision by ECHA

Developmental toxicity:

- Developmental toxicity in rat (OECD TG 414): NOAEL =>480 mg/kg bw

- A developmental toxicity in rabbits is imposed in a final decision by ECHA

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Two oral gavage studies of 28- and 90-day no fertility effects are seen at the highest dose tested of 500 and 1000 mg/kg bw.
An extended one-generation reproduction toxicity study will be performed in the near future.
Effect on fertility: via dermal route
Quality of whole database:
The dermal studies are insufficient for deriving information on systemic toxicity including fertility a summary of all repeated dose studies is presented below.
Additional information

Reproductive toxicity in the repeated dose toxicity 90 days in rat - oral

The safety of the test substance OTNE was examined in a subchronic (13 week) oral toxicity study in Wistar rats, according to OECD TG 408. OTNE was administered by daily oral gavage as a dilution in corn oil at levels of 0 (vehicle control), 30, 120 and 500 mg/kg body weight/day to groups of 10 rats/sex during 13 weeks. No relevant findings were seen on fertility and there were no histopathological lesions in reproductive organs. The increase of the relative testes weight at the mid dose only is not toxicologically relevant (absolute is more important than relative weight for this organ. Some dilatation of the uterus was reported, however as it this was also observed in the control group it was not considered an effect related to exposure to OTNE. Overall no effects on fertility were observed in the animals exposed to OTNE. The NOAEL for fertility is >=500 mg/kg bw,

Reproductive toxicity in the repeated dose toxicity 90 days in mouse and rat - dermal

The study was performed to assess the dermal repeated dose systemic toxicity as a result of dermal exposure to OTNE in the mouse and rat. The study was performed comparable to OECD TG 411, with some deviations (Dermal application without occlusion (not justified); partial oral exposure through grooming is expected, the top dose tested in mice was above the recommended dose)). Solutions of OTNE in ethanol were applied to the skin of mice and rats for 5 days per week for 3 months. There were 10 rodents in each dose group. Doses for which reproductive effects were evaluated were 25% and 50% OTNE in ethanol and 100% OTNE. These doses correspond approximately to 500 to 2000 mg/kg bw/day in mice and 125 to 500 mg/kg bw/day in rat. During the course of this study, samples were collected for estrous cycle characterization. At the end of the study, samples were collected for reproductive tissue evaluations. OTNE exposure via dermal application showed some minor but significant fewer sperm and lower sperm motility in the 2000 mg/kg bw/day male group. In females at 2000 mg/kg bw the estrus cycle was significantly extended at this dose with one day. None of these effects were observed in rats. The effects in mice are all observed at the high dose of 2000 mg/kg bw where very increased of relative liver weight was seen (up to 90%). Up to 1000 mg/kg bw no adverse effects on fertility are seen in male and female mice. The NOAEL for fertility is set at 1000 mg/kg bw.

Reproductive toxicity in repeated dose toxicity 28 days in rat - oral

This study was performed to assess the systemic toxicity of OTNE to the rat according to OECD TG 407. OTNE was administered by oral gavage, once daily, to three groups of rats for a minimum of twenty eight consecutive days, at dosage levels of 15, 150 or 1000 mg/kg/day. The test material was prepared as suspensions in corn oil at concentrations of 0.3, 3.0 or 20% w/v and was administered at a dosage volume of 5 ml/kg bw/day. After the 28-day exposure period, no effects on fertility were observed in the animals exposed to OTNE, therefore the NOAEL is set at >= 1000 mg/kg bw.

Effects on developmental toxicity

Description of key information

Prenatal development toxicity study (OECD TG 414): maternal NOAEL 240 mg/kg bw/day, developmental NOAEL 480 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 24, 2000 to Nov 24, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: US FDA “Guideline on detection of toxicity to reproduction for medicinal products” (1994; 1996)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Crl:CD®(SD)IGS BR VAF/Plus® rats weighed 218-246 g at study assignment and were about 65 days old at arrival. Pregnant animals were individually housed in stainless steel, wire-bottomed cages at a room temperature of 18-26C with a relative humidity of 30-70%, at least 10 air changes per hour, and a 12-hour light/dark cycle. Rats were given Certified Rodent Diet #5002 (PMI Nutrition International) and reverse osmosis water ad libitum.
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Rats were gavaged with 0 (control), 96, 240, or 480 mg/kg bw/day on gestation days 7-17 at dose volumes of 0.5, 0.1, 0.25, or 0.5 ml/kg bw, respectively. Doses were adjusted daily based on current body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (2 ml) of test substance were taken on first and last day of dosing for analysis. No further information available.
Details on mating procedure:
Virgin females were placed with males (1 female per male) for up to 5 days. Mating performance was evaluated daily and when spermatozoa in a vaginal smear and/or a copulatory plug were observed in females, the female was placed in individual housing and the day was designated as gestation day 0.
Duration of treatment / exposure:
Day 7-17 of gestation
Frequency of treatment:
Daily
Duration of test:
21 days
No. of animals per sex per dose:
25
Control animals:
other: 0.5 ml/kg bw/day of reverse osmosis deionized water (25 rats)
Details on study design:
Groups of pregnant rats (25/dose group) were gavaged with 0 (control), 96, 240, or 480 mg/kg bw/day on gestation days 7-17 at dosage volumes of 0.5, 0.1, 0.25, or 0.5 ml/kg bw, respectively. Viability (2x daily), abnormal clinical signs (daily), body weights (daily), abortions (daily), premature deliveries (daily), and feed consumption (gestation days 0, 7, 10, 12, 15, 18, and 21) were recorded. Mating performance was assessed. On gestation day 21, surviving rats were euthanized by carbon dioxide asphyxiation and Caesarean sections were performed to remove the foetuses.

Doses were selected based on a dose range-finding study:
Groups of pregnant rats (8/dose group) were gavaged with 0 (control), 240, 480, 960, and 1920 mg/kg bw/day on gestation days 7-17. On gestation day 21, all surviving rats were euthanized and examined for distribution of corpora lutea, implantation sites, and uterine content. Parental animals underwent gross necropsy and foetuses were weighed and examined. Body weights, body weight change, and feed consumption were generally comparable to those of controls throughout the study. Necropsy of the dams and examination of the litters revealed no differences in any of the test groups including controls. The recommended dose levels for the main developmental toxicity study were 240, 480, and 960 mg/kg bw/day. The no-observable-effect level (NOEL) for maternal and embryo-foetal toxicity was 240 mg/kg bw/day. Maternal toxicity with little or no developmental toxicity was reported at 960 mg/kg bw/day.
Maternal examinations:
Rats underwent gross necropsy of the thoracic, abdominal and pelvic viscera. Animals found dead or moribund were examined for gross lesions, pregnancy status, and uterine contents.
Ovaries and uterine content:
The number and distribution of corpora lutea, implantation sites, live and dead foetuses, and early and late resorptions were assessed. The uteri of apparently non-pregnant rats were examined to confirm absence of implantation sites.
Fetal examinations:
Live foetuses were euthanized with an intraperitoneal injection of Beuthanasia®-D Special. Sex and body weight of each foetus were recorded and any gross alterations were noted. About half of the foetuses were examined for soft tissue alterations and fixed in Bouin’s solution. Sections were kept in alcohol. The remaining foetuses were examined for skeletal alterations by staining with alizarin red S after evisceration and clearing.
Statistics:
Clinical observation and other proportion data were analysed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., weights, feed consumption, litter averages, etc.) were analysed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance when appropriate. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used. Count data from Caesarean sections were evaluated using the Kruskal-Wallis Test.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Two rats found dead and one delivered prematurely in the mid-dose group but these events were not considered related to test substance because they were not dose-related and 1 death was due to gavage error. Clinical signs included a statistically significant increase in the incidence of salivation in all treated groups compared to controls and a statistically significant increase in the incidence of urine-stained abdominal fur in high-dose animals compared to controls. Red, dried or red perioral substance was noted in all treated groups. Other clinical signs noted, but not considered related to the test substance, included localized alopecia, ptosis, soft or liquid faeces, chromodacryorrhea, ungroomed coat, red perivaginal substance and missing or broken incisors. Body weight gains were significantly reduced at all dose levels during gestation days 7-10 and throughout gestation at the highest dose. Body weights were significantly reduced on gestation days 16 and 17 at the highest dose. Feed consumption was significantly reduced in all dose groups during gestation days 7-12 and continued to be reduced throughout gestation at the highest dose only.
Key result
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Systemic effects
Description (incidence and severity):
Maternal effects were seen, see details on maternal toxic effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Foetal body weights were reduced, but not significantly, at the highest dose level. No effects attributable to test substance administration on any litter parameters (i.e, litter averages for corpora lutea, implantations, litter sizes, live foetuses, early and late resorptions, total and percent resorbed conceptuses per litter, the numbers of dams with any resorptions, dams with viable foetuses, dams with all conceptuses), gross external, soft tissue, or skeletal malformations, or number of ossification sites per foetus per litter at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 480 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Foetal body weight reductions (non-significant) seen only at maternally toxic doses.

Conclusions:
Based on persistent clinical observations and reduced body weights and feed consumption in the dams reported at the highest dose, the maternal NOAEL was considered to be 240 mg/kg bw/day. Reductions in foetal body weights were not statistically significant at the highest dose, and therefore, the developmental NOAEL was considered to be 480 mg/kg bw/day. It was concluded that OTNE was not a developmental toxicant.
Executive summary:

A developmental toxicity study was performed in accordance with the US FDA Guideline on detection of toxicity to reproduction for medicinal products, which is equivalent to OECD414. The study was performed under GLP conditions.

Groups of pregnant rats (25/dose group) were gavaged with 0 (control), 96, 240, or 480 mg/kg bw/day on gestation days 7-17 at dosage volumes of 0.5, 0.1, 0.25, or 0.5 ml/kg bw, respectively. Viability, abnormal clinical signs, body weights, abortions, premature deliveries, and feed consumption were recorded. Mating performance was assessed. On gestation day 21, surviving rats were euthanized by carbon dioxide asphyxiation and Caesarean sections were performed to remove the foetuses. Rats underwent gross necropsy. Foetuses were assessed for litter parameters and gross external, soft tissue, or skeletal malformations.

Maternal animals showed reduced body weights and persistent clinical signs (e.g., increased salivation and urine-stained abdominal fur) at the highest dose. There were no treatment-related effects on developmental parameters at any dose, although reduction (not statistically significant) in foetal body weight was noted at the highest dose. Based on these results, the maternal NOAEL was 240 mg/kg bw/day and the developmental NOAEL was 480 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
480 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is high because in two different tests fertility has been investigated: the developmental toxicity study and a study in which the transfer across the placenta and into milk of rats during and after pregnancy following repeated oral administration was studied. These studies adequately fulfill the REACH requirements.
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

Rat: A developmental toxicity study was performed in accordance with the US FDA Guideline on detection of toxicity to reproduction for medicinal products, which is equivalent to OECD TG 414. The study was performed under GLP conditions. Groups of pregnant rats (25/dose group) were gavaged with 0 (control), 96, 240, or 480 mg/kg bw/day on gestation days 7-17 at dosage volumes of 0.5, 0.1, 0.25, or 0.5 ml/kg bw, respectively. Viability, abnormal clinical signs, body weights, abortions, premature deliveries, and feed consumption were recorded. Mating performance was assessed. On gestation day 21, surviving rats were euthanized by carbon dioxide asphyxiation and Caesarean sections were performed to remove the foetuses. Rats underwent gross necropsy. Foetuses were assessed for litter parameters and gross external, soft tissue, or skeletal malformations. Maternal animals showed reduced body weights and persistent clinical signs (e.g., increased salivation and urine-stained abdominal fur) at the highest dose. There were no treatment-related effects on developmental parameters at any dose, although reduction (not statistically significant) in foetal body weight was noted at the highest dose. Based on these results, the maternal NOAEL was 240 mg/kg bw/day and the developmental NOAEL was 480 mg/kg bw/day.

Rabbit: A pre-natal developmental toxicity study on a second species is required according to Annex X, Section 8.7.2. as a standard information requirement, in addition to a prenatal developmental toxicity study in a first species according to Annex IX, Section 8.7.2. To fulfill this endpoint a pre-natal developmental toxicity study (Annex X, Section 8.7.2.; test method: EU B.31./OECD TG 414) in a second species (rabbits), oral route, is planned.

Justification for classification or non-classification

Based on the results, which indicate no reproduction and developmental toxicity effects of OTNE, the substance does not need to be classified as toxic to reproduction according to EU CLP Regulation (EC 1272/2008 and its amendments).