Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

- OECD 443; GLP; Wistar rats; oral gavage; 10, 30, 100 mg/kg; NOAEL (reproduction) >/=100 mg/kg

- OECD 422; GLP; Wistar rats; oral gavage; 40, 125, 375 mg/kg; NOAEL (reproduction) >/= 375 mg/kg

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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 investigated of possible pre- and postnatal effects on development. In addition, a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats was performed (OECD 443, 2019).

Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.

In addition, the study provided and/or confirmed information about the effects of the test substance on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behaviour, conception, pregnancy, parturition, and lactation.

Furthermore, the information obtained from the assessments characterized potential effects in those systems. The dose levels in this study were selected to be 0, 10, 30, 100 mg/kg/day, based on the results of a preliminary reproductive toxicity study (combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422) with oral exposure of the test substance in rats. In this preliminary study, animals were dosed with 0, 40, 125 and 375 mg/kg/day. From dose 125 mg/kg/day, adverse findings observed were ulcers and inflammation of the forestomach in males and hyperplasia squamous cells in males and females. Other test item-related findings consisted of an increase in liver weights in males and females and an increase in hyaline droplet accumulation in the male kidneys at 375 mg/kg/day. Based on the severe findings in the stomach, a parental local NOAEL was established of 40 mg/kg/day in this study. As animals are dosed for a longer time period for the Extended One-Generation Reproductive Toxicity Study (e.g. F0-males 11-13 weeks versus 4 weeks in an OECD 422 study), a maximum dose level of 100 mg/kg/day was selected. Further doses selected were 10 mg/kg bw/day and 30 mg/kg bw/day. For the F0-generation, the following parameters and end points were evaluated in this study:

mortality/ moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. In addition, functional observations were evaluated in females in this study.

For the F1-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial Separation, day of first estrus, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones). No parental toxicity was observed up to the highest dose level tested (100 mg/kg/day). At 30 and 100 mg/kg/day, a minor increase in sodium was noted in males and additionally, at 100 mg/kg/day, urea and potassium levels were slightly increased. As values were only slightly above the range considered normal (sodium) or remained within normal range (urea and potassium) and no corroborative microscopic findings were found, these changes were considered non-adverse.

No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, functional observations (females), body weight, food consumption, heamatology, coagulation, thyroid hormone analysis, urinalysis, macroscopic examination, organ weights and microscopic examination). No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg/day). No test item-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, sperm analysis, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the pre-weaning phase.

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, duration of gestation, viability and weaning indices, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), and macroscopic examination). No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day) during the post-weaning phase.

At 100 mg/kg/day, increases in neutrophil and lymphocyte levels were found in males. As values remained within normal range and no corrobative microscopic findings were found, these changes were considered non-adverse.

No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, body weight, food consumption, coagulation, clinical chemistry, thyroid hormone analysis, urinalysis, splenic lymphocyte subpopulation, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, sperm analysis, macroscopic examination, organ weights, ovarian follicle and corpora lutea counts, and microscopic examination, including stagedependent qualitative evaluation of spermatogenesis in the testis).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1), the no-observed-adverse-effect levels (NOAEL) of the test substance for general toxicity (F0 and F1), reproduction, and development was observed to be at least 100 mg/kg bw/day.

Additionally Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP (2018). The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Estrous cycles examinations, reproductive organ weight assessment and respective histopathology was performed. For the testes of all selected males of the lowest and highest exposure level, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). There were 2/10 control group couples, 1/10 couples treated at 40 mg/kg/day, 3/10 couples treated at 125 mg/kg/day and 1/10 couples treated at 375 mg/kg/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. The length and regularity of the estrous cycle were not considered to have been affected by treatment. Moreover, the mating index, precoital time, number of implantation sites and the fertility index were unaffceted by treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no parental reproductive toxicity was observed up to and including the highest tested dose of 375 mg test item/kg bw/d due to the abscence of respective adverse effects under the conditions of this study.

Effects on developmental toxicity

Description of key information

- OECD 414; GLP; New Zealand White rabbits; oral gavage; 50, 150, 450 mg/kg; NOAEL (development) 450 mg/kg bw/day

- equivalent to OECD 414; Sprague-Dawley rat; oral gavage; NOAEL (development) >/= 250 mg/kg bw/day

- OECD 422; GLP; Wistar rats; oral gavage; 40, 125, 375 mg/kg; NOAEL (development) >/= 375 mg/kg

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Jun 2018 - 25 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 15-21 weeks old
- Weight at study initiation: between 3043 g and 4268 g
- Housing: housed individually in cages with perforated floors (Ebeco, Germany)
- Diet: ad libitum, Pelleted diet for rabbits (Global Diet 2030 from Harlan)
- Water: ad libitum, Municipal tap water
- Acclimation period: least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21
- Humidity (%): 38 to 97
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension; protected from light. If not used on the same day of preparation, the aliquots were stored in the refrigerator set to maintain 4°C for a maximum of 8 days. On the day of use, they were removed from the refrigerator and allowed to warm to room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing.m Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: 1% Aqueous carboxymethyl cellulose with 0.5% Tween 80
Analytical verification of doses or concentrations:
yes
Remarks:
Acquity UPLC system
Details on analytical verification of doses or concentrations:
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The females arrived on Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
from Day 6 to Day 28 post-coitum
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the tolerability study and dose range finder and in an attempt to produce graded responses to the test item. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity including mortality was observed at 750 mg/kg. Therefore, the dose levels selected in the current main teratology study were 0, 50, 150 and 450 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
All animals (including female no. 75 euthanized before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. In addition, the gastro-intestinal tract was examined for signs of irritation/corrosion; the oesophagus was examined internally. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No tissues, except the uterus, were weighed. Each ovary and uterine horn of all animals was dissected and examined as quickly as possible.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]

- Soft tissue examinations: Yes: [all per litter]

- Skeletal examinations: Yes: [all per litter]
Subsequently, the skeletal examination was done on all fetuses from all groups from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

- Head examinations: Yes: [half per litter]
Statistics:
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
see table 1
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicological relevant findings up to 450 mg/kg.
Reduced faeces production (up to a moderate degree) was observed in 14, 18, 19 and 18 females in the control, 50, 150 and 450 mg/kg groups, respectively, on several days during treatment. As all groups were affected (including the vehicle control group) and in the absence of a dose-related trend, no toxicological significance was attached to this observation. Incidental findings of note were red fluid on the manure tray in one high dose female, and lean appearance and/or piloerection in another high dose female. These findings were considered not to be toxicological relevant as they were observed in the vehicle control group at the same incidence (red fluid on the manure tray, lean appearance), were transient (lean appearance and piloerection) and all gravid females in this study had a normal pregnancy.
One female in Group 3 was noted with missing toes (two missing toes on the right foreleg and 1 missing toe on the left foreleg; taken from the study daybook). This finding was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain, and/or were observed in control females only. They were therefore of no toxicologically relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatmentwith the test item.
One female from the high dose group had to be euthanized after 4 days of treatment. Immediately after dosing on post-coitum Day 9, she was observed with respiratory problems (gasping) and moribund. At necropsy, red foamy content of the trachea, several reddish foci in left caudal lobe of the lungs and grey-white discolouration of the left caudal lobe of the lungs were noted. Taken together, these findings were indicative of complications during the oral gavage procedure. Therefore, the preterm sacrifice of this female was considered unrelated to the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight, body weight gain before and after correction for (gravid) uterus weight of treated Group 2, 3 and 4 females were unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded up to 450 mg/kg. In the high and mid dose group each, a trend towards slightly lower absolute and relative food consumption compared to the concurrent control group was noted from Days 6-15 and Days 12-15 post-coitum, respectively. As changes were only slight (reaching no statistical significance) and transient (complete recovery was seen from Days 15-18 post-coitum onwards), it was not considered as adverse.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Moreover, the examination of the gastro-intestinal tract and the internal examination of the oesophagus did not reveal any signs of irritation and/or corrosion. The observation of missing toes in one Group 3 female was considered to be a congenital abnormality and thus unrelated to treatment with the test item. Remaining incidental findings among control and treated animals included watery-clear cysts of the oviducts, alopecia, scabs or an isolated wound. These findings are occasionally seen among rabbits used in these types of study, and in the absence of a dose relationship they were considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Overall, the number of pregnant females, corpora lutea and implantation sites, and pre-and post-implantation loss in the control and treatment groups were considered to be unaffected by treatment. Mean pre-implantation loss was 3.6, 3.0, 3.0 and 10.5% for the control, 50, 150 and 450 mg/kg groups, respectively. The higher pre-implantation loss observed in the 450 mg/kg group could largely be attributed to one female (no. 76) who had 11 corpora lutea, but only 3 implantation sites, resulting in a pre-implantation loss of 72.7%. After excluding this female pre-implantation loss was 7.2%. As all values remained within the available historical control range the slightly higher pre-implantation loss in the high dose group was considered unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificed in extremis on Days 4 and 7 of treatment at 750 mg/kg.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 450 mg/kg.
Mean combined (male and female) fetal body weights were 37.8, 38.2, 39.1 and 38.0 gram for the control, 50, 150 and 450 mg/kg groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 450 mg/kg.
Mean sex ratios (males:females) were 46:54, 50:50, 44:56 and 56:44 for the control, 50, 150 and 450 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.8, 10.1 and 9.3 fetuses/litter for the control, 50, 150 and 450 mg/kg groups, respectively. As all values remained within the available historical control range the slightly lower litter size in the high dose group was considered unrelated to treatment.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 450 mg/kg/day.
Two external malformations were observed in this study. Two fetus of Groups 2 and 4, respectively, both had an open eye with relating findings of the eye/lens noted at soft tissue cephalic examination (i.e. eyelids not joined, lens attached to cornea and/or lens misshapen). Due to the single occurrence of this malformation in a low and high dose fetus, it was considered to be chance finding. A flexure of the carpal and/or tarsal was the other malformation and this occurred in the control group in one fetus (both tarsals, without apparent skeletal origin) and in late resorptions in two fetuses (one or both carpals, not skeletally examined). Because carpal and/or tarsal flexures were observed at the control level only, this was considered spontaneous in origin. External variations were not observed in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment at 450 mg/kg/day.
The only fetus with a malformation was one fetus of the high dose, which had a vertebral anomaly with associated rib anomaly. A vertebral anomaly is the most common skeletal malformation among historical control fetuses and at the single occurrence noted in this study, it is considered spontaneous in origin. A statistical significant increase in the litter incidence of fetuses with caudal shift of pelvic girdle was observed in the high dose group. This variation occurred at an incidence of 21.5%
versus 8.7% per litter in the concurrent control group. Because the litter incidence of caudal shift of pelvic girdle was well within the historical control data range (mean 16.5% per litter; P5 – P95: 2.8 - 34.5% per litter), the higher incidence of this finding in Group 4 was considered to have occurred by chance and not to be toxicologically relevant. All other variations noted were not considered treatment related as they occurred infrequently, at frequencies that were within the range of available historical control data, or were observed in control fetuses only.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 450 mg/kg/day. Visceral malformations occurred in 0 (0), 1 (1), 4 (3) and 3 (2) fetuses (litters) in the control, 50, 150 and 450 mg/kg groups, respectively.
In Group 4, the three malformed foetuses (out of two litters) had a malpositioned testis. This malformation was also noted in Group 3 fetus, but not in fetuses at the control and low dose levels. Nevertheless, because a malpositioned testis is one of the most common visceral malformations in historical control fetuses, this was considered not to be treatment related. The other malformed Group 3 fetuses all had a multiple cardiovascular malformation. As no cases occurred in Group 4 and none of
the individual malformations was uncommon among historical control fetuses these were considered not toxicologically relevant. The affected fetus in Group 2 had a diverticulum of the intestine, which at the single occurrence at the low dose was considered spontaneous in origin.
All variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar- Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed on August 2019
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ReachCentrum SA
- Batch number of test material: 180003P040
- Expiration date of the lot/batch: 31. Dec 2018
- Purity/Composition: 100% (UVCB)
- Appearance: clear, colorless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under storage conditions: until 31. Dec 2018
- Stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, stability for at least 8 days in
the refrigerator, and stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at
appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han), outbred, SPF-Quality
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males), 13 weeks (females)
- Weight at study initiation: males: 251- 322 g; females: 198- 263 g
- Housing: individually (females during the post-mating phase and lactation phase with the pups) and grouping (pretest, males during the post-amting phase
- Diet: ad libitum; pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum
- Acclimation period: for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40-70 (daily mean relative humidity of 36 to 60%)
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure. Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 d
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single housing
Duration of treatment / exposure:
males: 29 days; females: 50-62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and the duration of pregnancy and at least 14 days after delivery,
up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 40-53 days.
Frequency of treatment:
daily (7d/week)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- in total 10 animals/ sex/ dose
- 5 animals /sex/ dose were selected for functional tests (males only), clinical pathology, collection of full list of organs/tissues at macroscopic examination, organ weights (full list) and histopathology (full list)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substance was orally (gavage) applied at concentrations of 0, 557 and 1855 mg/kg body weight per day for 14 days to four animals per test group and sex. No treatment related alterations were observed in any of the observed parameters at 1855 mg/kg body weight (high dose) dose level. Hence, this short-term repeated dose oral toxicity study was performed at similar dose levels.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: First day of treatment (prior to dosing) and weekly thereafter (after dosing)
- Body Weight Gains: Calculated against the body weight on Day 1 (premating, mating and lactation periods) or Day 0 (postcoitum period).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule for examinations: quantitatively 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.
- Relative Food Consumption Calculated against the body weight for scheduled intervals.

WATER CONSUMPTION: not quantitative
- Subjective appraisal was maintained during the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Females which delivered on PND 14-16, females which failed to deliver post-coitum Day 25 or 26 (with evidence of mating) or 25 days after the last day of the mating period (without evidence of mating), dams with no surviving pups were euthanized within 24 hours after the last pup was found dead or missing

- Organs examined:
Selected animals: Tissues identified in table 5 (see any other information on materials and methods) with the exception of animal identification, aorta, nasopharynx, esophagus, harderian
gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue,
Females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina,
Females with total litter loss: Mammary gland,
Remaining animals: Gross lesions/masses.

OTHER: Hematology, coagulation, clinical chemistry, thyroid hormones (see any other information on materials and methods)





Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No (exception: if no macroscopic visible implantation sites were present)
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
Gestation index, postimplantation survival index, live birth index, viability index, lactation index (for detailed formulas see "Any other information on materials and methods"
Historical control data:
Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: Maternal body weight gain (period 2015- end 2017), clinical chemistry (period 2015- end 2017), thyroid hormone analyses (period 2015- end 2017), post-implantation survival index (period 2015 - May 2018)


Clinical signs:
no effects observed
Description (incidence and severity):
Salivation seen after dosing among animals of all dose groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its
time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity
Mortality:
no mortality observed
Description (incidence):
One female of the control group (no. 49) was euthanized on Lactation Day 1, as she had a total litter loss (at first litter check she had only dead pups).
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant change in relative food consumption in 40 mg/kg females during post-coitum Days 17-20 was considered to be unrelated to treatment, since no trend was
apparent regarding dose and duration of treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A decreased number of reticulocytes was observed in females at 125 mg/kg, which was considered unrelated to administration of the test item due to absence of a dose-related trend response.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related irregular surface was observed in the (fore)stomach in 2/10 females treated at 375 mg/kg/day.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Watery fluid in the uterus, found in one control female and 3 females treated with 125 mg/kg, is related to a stage in the estrous cycle and is a normal finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the (fore)stomach (2/5females). Squamous cell hyperplasia was present in females at 375 mg/kg/day up to moderate degree. This correlated with the macroscopic irregular surface.





Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125
and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- No deficiencies in maternal care were observed.
- Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse developmental effects observed up to and including the highest dose tested.
Fetal body weight changes:
not examined
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Dead pubs at the first litter check: 11 in the control group and 1 daed pub in the exposure group 40 mg/kg bw/d
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
One litter each was affected in the control, 125 and 375 mg/kg bw/d group (one dead pub), representing 1, 1.2 and 1% of living pubs
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- The post-implantation survival index, the live birth index, viability index and lactation index were not affected by the treatment.
- No clinical signs occurred among pups that were considered to be related to treatment. For the pup of female no. 71 (375 mg/kg) who was missing on Day 4, less milk in the
stomach and a lean appearance were noted on Day 1. Alopecia of the whole body was noted for several pups of the control, 40 and 375 mg/kg groups (not toxicological relevant as within the range considered normal)
- No macroscopic findings among the pubs
Dose descriptor:
NOAEL
Effect level:
> 375 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse developmental effects observed up to and including the highest dose tested.
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1 Developmental indeces

Dose in mg/kg bw/d

0

40

125

375

Post-implantation survival index (%)

92

79

88

85

Live birth index (%)

90

99

100

100

Viability index (%)

99

100

99

99

Lactation index (%)

100

100

100

100

Table 2 Developmental data

Dose in mg/kg bw/d

0

40

125

375

Total number of offspring born

109

84

85

104

Total number of uterine implantation sites

118

106

97

123

Number of live offspring on day 1 after littering

98

83

85

104

Number of live offspring on day 4 (before culling)

97

83

84

103

Number of live offspring on day 4 (after culling)

63

65

56

70

Number of live offspring on day 13 after littering

63

65

56

70

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed no adverse signs of developmental toxicity in in maternal animals and their offspring up to and including the tested dose of 375 mg/kg bw/d.

Therefore, the NOAEL for developmental toxicity was set to be at least 375 mg/kg bw/d.
Executive summary:

Wistar Han rats were treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg according to OECD 422 and in compliance with GLP. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

The following developmental parameters were determined: Number of implantation sites, gestation index and duration, parturition and maternal care, live birth index, viability index and lactation index, post-implantation survival index and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)).

Gestation index and duration of gestation were not considered to be affected by treatment. Except for one female of the control group, all pregnant females had live offspring. The gestation indices were 89% for the control and 100% for the 40, 125 and 375 mg/kg groups, respectively. 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. The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92%, 79%, 88% and 85% for the control, 40, 125 and 375 mg/kg groups, respectively. The slightly lower post-implantation survival index for the 40 mg/kg group was considered the result of the low litter size of female no. 51 which only had one pup, but 10 implantation sites. The survival indices for all other groups were considered within normal range. Litter size was not considered affected by treatment. Live litter sizes were 10.9, 9.2, 12.1 and 11.6 living pups/litter for the control, 40, 125 and 375 mg/kg groups, respectively. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 90%, 99%, 100% and 100% for the control, 40, 125 and 375 mg/kg groups, respectively.

Eleven pups of the control group and one pup at 40 mg/kg were found dead at first litter check. Eight of these pups were of the control group female, which had a total litter loss. No toxicological relevance was attributed to these dead pups since the mortality incidence mainly occurred in the control group and did not show a dose-related trend. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 99%, 100%, 99% and 99% for the control, 40, 125 and 375 mg/kg groups, respectively. Three dead/missing pups were present throughout the study (one pub of the control, 125 and 375 mg/kg bw/d expsoure group), however, the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. 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, resulting in a lactation index of 100% for all groups. The postnatal pub deveopment was unaffected by the treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no developmental toxicity was observed up to and including the highest tested dose of 375 mg/kg test item due to the abscence of respective adverse effects under the conditions of this study.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1982 - Jun 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The test substance was administered by gavage to 22 pregnant rats from day 6 to day 15 of gestation to evaluate the embryo/fetal toxicity and
terratogenic effects.
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
- Product name: C-258
- Lot No.: 3-81
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 5 weeks upon receipt, ca. 14 weeks at start of mating period
- Weight at study initiation: females ca. 233 g on day 0
- Housing: individually, 1 male with 2 feamles during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.5 °C )76°F (1.0 S.D.)
- Humidity (%): 57% (4.8 S.D.)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was prepared fresh weekly by mixing a specified amount of the test material with the
vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): none given
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot No.: 80235
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: maximum of 3 weeks
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no data]
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
days 6 through 15 of gestation
Frequency of treatment:
once daily
Duration of test:
animals were sacrificed on day 20 of gestation
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 female animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: mortality at 500mg/kg, for details see supporting study
- Rationale for animal assignment: using a table of random permutations
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily


BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 9, 12, 15 and 20 of gestation


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus and ovaries of each dam
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter ]
- Skeletal examinations: Yes: [2/3 per litter ]
- Head examinations: No data
Statistics:
various statistical methods used: National Cancer Institue Package, Box's test for homogeneity of variances, one-way classification analysis of variance
(ANOVA), Dunnett's T-test, one degree of freedom Chi-square test with Yates continuity correction, Fisher's Exact test. All pairwise comparisons were
evaluated at the 5.0% probability (one-tailed) level.
Indices:
no data
Historical control data:
no data
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hunched posture (3), soft feces (2 pregnant females and the one non-pregnant female), thin (1), bloody crust around nose, mouth, eyes, front paws (1), sores around head (1), and alopecia on the back (1) occured only in single animals - exact number given in parenthesis - and were likely caused by the irritant properties of the test substance. These signs were not considered indicative of systemic toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 out of 22 animals died, but without apparent relation to treatment.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Red areas (all lobes) in the lung, a dilated pelvis in the kidney, uterus contained implantation sites, ovaries contianed corpora lutea, small and cloudy eyes were observed among two females. Twenty out of 22 females had no observable gross pathological findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses observed.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Compared to control group, no differences were observed.
Other effects:
no effects observed
Description (incidence and severity):
- Mean number of corpora lutea showed no adverse effects
- Mean number of implantations and mean implantation efficiency (number of implantations per number of corpora lutea) comparable to control
- No changes in uterine weights (gravid and non-gravid)
Details on maternal toxic effects:
One female did not become pregnant.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Cleft palate and small lower jaw in one pup
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No anomalies were observed. Sixty-two fetuses with variants across 19 litters were observed. Incomplete ossification in skull, vertebrae, and pelvis, small supraoccipital in skull irregular osification in rib cage, nonfused centra vertebrae, less than three ossified caudals (vertebrae), and small pubis.
Visceral malformations:
no effects observed
Description (incidence and severity):
Four fetuses showed variants. Two showed dilated renal pelves, one had a dilated ureter and one was small (small fetus). However, the incidence was low and was considered not to be related to treatment. In addition, findings were comparable to control group.
Other effects:
no effects observed
Description (incidence and severity):
- No differences observed in crown-rump distance in males and females compared to control
Details on embryotoxic / teratogenic effects:
172 fetuses were examined and 168 fetuses appeared normal at gross pathology. Compared to control (128 examined, 114 appeared normal), no differences were identified.

Details on embryotoxic / teratogenic effects:
No statistically significant effects compared to controls were observed. A single pub was diagnosed with cleft palate, and situs inversus was observed in one control pub. Due to the low incidence, these finding are considered incidental. There were no visceral abnormalities. The number of visceral variations was comparable between control and treatment groups (62 of 172 pubs and 73 of 191 pubs, respectively). Sex distribution, fetal viability, fetal body weights and uterine weights were also unaltered by treatment.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
At the dose level investigated in this study, 250 mg/kg, no adverse effects on maternal systemic toxicity and on maternal and fetal developmental toxicity.
Executive summary:

This report presents the results of a study designed to evaluate the embryo/fetal toxicity and teratogenic effects of a series of ten experimental compounds (C-l80, C-181, C-182, C-236, C-254, C-255, C-256, C-257, C-258, and C-259) when administered by gavage to pregnant rats from Days 6 through 15 of gestation. Each test material was administered

to a group of twenty-two female rats at a single dose level specific to that material (Groups 2 through 12). An eleventh group (Group 1) served as a common control and received only corn oil.

Maternal, ovarian, uterine, litter, and fetal data were evaluated for evidence of compound effects.

For the test substance C-258, no adverse effects were observed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
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 investigated in a teratogenicity study according to OECD 414 and in compliance with GLP (2019). The objectives of this study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.The dose levels in this study were selected to be 0, 50, 150, 450 mg/kg/day, based on the results of the dose range finder and tolerability study. In the dose range finder, no dose-limiting toxicity was noted up to 400 mg/kg. However, in the tolerability study severe toxicity was observed at 750 mg/kg.

The following parameters and end points were evaluated in this study for the F0-generation:

mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents, and maternal pregnancy data.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. No maternal toxicity was observed in the 50, 150 and 450 mg/kg groups.

No developmental toxicity was observed in the 50, 150 and 450 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance of at least 450 mg/kg was established.

Higher doses were not tolerated as two out of three females in the tolerability study had to be sacrificedin extremis on Days 4 and 7 of treatment at 750 mg/kg.

In a supporting teratology screening study comparable to OECD 414, the test substance was administered by gavage to 22 pregnant rats from day 6 to day 15 of gestation at a dose level of 250 mg/kg bw/d to evaluate the embryo/fetal toxicity and teratogenic effects (Hazleton Labs Inc., 1983, Val. 2). As determined in a previous maternal tolerance study, at 500 mg/kg pathological findings in stomach, liver and lung are described, in additon 500mg/kg led to mortality in one of six animals. The dose level for the main study was thus set at one half of the lethal dose. One out of 22 animals died, but without apparent relation to treatment. Clinical signs such as rough haircoat (3), hunched posture (2), bloody crusts on nose, mouth, eyes, and/or front paws (1) occurred only in single animals - exact number given in brackets - and were likely caused by the irritant properties of the test substance. These signs were not considered indicative of systemic toxicity. No significant differences in body weight and food and water consumption were noted compared to controls.

There were no differences in the number of corpora lutea, implantation sites, and pre- and post-implantation loss. Sex distribution, fetal viability, fetal body weights and uterine weights were also unaltered by treatment. There was no treatment related increase in gross malformation and visceral abnormalities or variations.

As a consequence, the NOAEL for maternal toxicity is at least 250 mg/kg bw/day and the NOAEL for embryotoxicity/teratogenicity is also at least 250 mg/kg bw/day.

In addition no indications of a possible developmental toxic effect were observed in a recent OECD 422 study in Wistar Han rats treated with the test item by daily oral gavage at dose levels of 40, 125 and 375 mg/kg (2018). The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-62 days). Females that failed to deliver pups were treated for 40-53 days.

Estrous cycles examinations, reproductive organ weight assessment and respective histopathology was performed. For the testes of all selected males of the lowest and highest exposure level, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). There were 2/10 control group couples, 1/10 couples treated at 40 mg/kg/day, 3/10 couples treated at 125 mg/kg/day and 1/10 couples treated at 375 mg/kg/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. The length and regularity of the estrous cycle were not considered to have been affected by treatment. Moreover, the mating index, precoital time, number of implantation sites and the fertility index were unaffceted by treatment.

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, no parental reproductive toxicity was observed up to and including the highest tested dose of 375 mg test item/kg bw/d due to the abscence of respective adverse effects under the conditions of this study.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for reproductive toxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) 2018/1480 of 4 October 2018.