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Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the availability of a prenatal toxicity study, the performance of a screening study is waived. As no adverse effects were observed on reproductive (and developmental) parameters, performance of an EOGRTS is considered not scientifically justified.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
Reason / purpose for cross-reference:
data waiving: supporting information
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Quality of whole database:
good
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 results of a 90-day repeated dose toxicity study with FDCA demonstrated that the repeated dose toxicity is at least 1000 mg/kg bw/day. No effects on reproductive organs were observed up to the highest dose tested. Literature search on reproduction toxicity of FDCA and 20 related structures did not demonstrate reproductive toxicity below the limit values based on repeated dose toxicity. In the absence of general toxicity for FDCA up to and including 1000 mg/kg bw/day in a sub-chronic toxicity study, and structural related substances show no significant reproductive toxicity, from scientific and ethical point of view it is not justified to perform further reproductive toxicity studies with FDCA. 

Effects on developmental toxicity

Description of key information

Based on the results of a Prenatal Developmental Toxicity Study in rats performed according to OECD/EC guidelines and GLP principles it is concluded that the NOAEL (maternal and developmental) for FDCA is >=1000 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:
29 June 2020 - 16 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The test was initiated after ECHA decision was received.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required

pH: 2.4-2.5 at concentration of 0.1wt%
Stability in vehicle:
- Water: Stable
- 1% Carboxymethyl cellulose: Stability for at least 6 hours at room temperature under normal laboratory light conditions, and for at least 8 days in the refrigerator (2-8°C) has been confirmed over the concentration range 1 to 200 mg/mL (suspension), Charles River Study No. 20247702.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 178 - 280 g
- Fasting period before study: no
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. For psychological/environmental enrichment and nesting material, animals were provided with paper and aspen wooden sticks.
- Water: Municipal tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24 (target), 21 (actual daily mean)
- Humidity (%): 40-70 (target), 54-77 (actual daily mean)
- Air changes (per hr): ten or more (no air circulation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Sep 2020 To: 01 Oct 2020
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous solution
Details on exposure:
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 for maximally 8 days in advance as daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator.
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. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

- Dose volume: 5 mL/ kg bw
- Dose concentrations: 0, 20, 60 and 200 mg/mL for the control low, mid and high dose group, respectively
- The dose volume for each animal was based on the most recent body weight measurement.
- The doses were given using a plastic feeding tube.
- The dosing formulations were stirred continuously during dose administration.
- A dose control system was used as additional check to verify the dosing procedure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for concentration analysis from all groups (sample collected from approximately the middle) and for homogeneity analysis from the low and the high dose groups (sample collected from approximately top, middle and bottom) in week 1.
Analyses were performed using a validated analytical procedure (Charles River Study
No. 20247702).
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 ±15% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability analyses were performed previously as part of a 90-Day Toxicity Study with FDCA by
oral gavage in rats (Charles River Study No. 501479). These results demonstrated that the test
item is stable in the vehicle when prepared and stored under the same conditions at
concentrations bracketing those used in the present study.
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum at the test facility.
Duration of treatment / exposure:
15 days (from Day 6 to 20 post-coitum, inclusive)
Frequency of treatment:
Once daily, 7 days a week
Duration of test:
16 days
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
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 range finder in the rat and in an attempt to produce graded responses to the test item. Time-mated female Wistar Han rats were received and test item and vehicle were administered to the appropriate animals by once daily oral gavage from Day 6 to Day 20 post-coitum, inclusive. Three groups were included: a control group, a group dosed at 500 mg/kg bw/day and a group dosed at 1000 mg/kg bw/day. Each group consisted of 5 females. The dose levels were selected based on the results of a 90-Day Toxicity Study with FDCA by oral gavage in rats (Charles River Study No. 501479) in which no adverse effects were recorded up to and including a dose of 1000 mg/kg bw/day. In-life procedures, observations, and measurements in the dose range finder were identical as for the main study, except for anogenital distance measurements and blood collection for thyroid hormone analysis, which were not conducted. Terminal procedures of F0-animals were identical as for the main study, except for thyroid weight measurements and fixation of the thyroid, which were not conducted. Each viable fetus of animals surviving to planned necropsy was externally examined in detail, weighed and sexed. All live fetuses were euthanized by decapitation. No visceral (internal) or skeletal examination was performed. No fetuses and late resorptions were observed with malformations and were therefore discarded.
The dose range finder did not reveal signs of toxicity (details given below in the results section).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, starting on Day 2 post-coitum onwards up to the day prior to necropsy
- detailed clinical observation was performed weekly (including removal of animals from the cages), beginning during the Pretreatment Period, and on the day of necropsy
- Cage debris was examined to detect premature birth

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-
21 post-coitum

WATER CONSUMPTION: Yes
- 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 21 (by a carbon dioxide inhalation (gradual fill) procedure)
- All animals were subjected to an external, thoracic and abdominal examination, with special
attention being paid to the reproductive organs. All macroscopic abnormalities were recorded,
collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No organs (except for the gravid uterus and thyroid gland) were weighed.
- Thyroid glands of all animals were histopathologically examined.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the uterus
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of early and late resorptions.
- The sex of each fetus based on the anogenital distance.
In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Blood sampling:
Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected between 7:00 and 9:00 a.m. from the jugular vein. Blood samples were processed for serum, and serum was analyzed for Triiodothyronine (T3), Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH).
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity
using a small metal feeding tube. For one fetus this was not possible due to a malformation, this fetus was euthanized by instrascapular injection of sodium pentobarbital.

Litters were subjected to detailed external, visceral and skeletal examinations.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External: Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and
weighed. For late resorptions, a gross external examination was performed.

Visceral: The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and
dissected using a technique described by Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations (2 fetuses) were stored in 10% formalin. Any remaining tissues (from the fetuses used for fresh visceral examination) were discarded.

Skeletal:
All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in
potassium hydroxide. Thereafter, they were stained with Alizarin Red S by a method similar
to that described by Dawson. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. The following pairwise comparisons were made: Low dose group vs. control group, mid dose group vs. control group, high dose group vs. control group. Analyses were performed according to the statistical matrix included below when possible but exclude any group with less than 3 observations.
Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were
conducted using Dunnett’s test.
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Historical control data:
Historical control data were included in the report where relevant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing for one animal at 300 mg/kg bw/day and two animals at 1000 mg/kg bw/day was considered not 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 rather than a sign of systemic toxicity.
Abnormal breathing sounds was recorded for one animal at 1000 mg/kg bw/day, which was
regarded to be unrelated to treatment with the test item due to the incidental occurrence. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus were
unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Slightly lower food consumption levels (not statistically significant) were observed for most treatment intervals at 1000 mg/kg bw/day. These changes were not toxicologically relevant as the changes in food consumption were considered slight (0.95-0.92x of control).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total triiodothyronine (Total T3), total thyroxine (Total T4) and thyroid stimulating hormone (TSH) were unaffected by treatment up to 1000 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item up to 1000 mg/kg bw/day.
Incidental findings among control and treated animals included dark red discoloration and enlargement of the mandibular salivary gland, abnormal appearance and hernia of the medial
lobe of the liver, abnormal appearance of the abdominal wall and scab of the skin and dark red discoloration and foci of the thymus. These findings are occasionally seen among rats used in these types of study and in the absence of correlated microscopic findings they were considered changes of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites, pre- and postimplantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 3 females were not pregnant (two controls and one female in the 100 mg/kg bw/day group), i.e. the animals had no corpora lutea and no implantation sites. The isolated case of nonpregnancy at 100 mg/kg bw/day was unrelated to treatment with the test item as the incidence of non-pregnancy was within normal range (as was evident from the two non-pregnant females in the control group).
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed up to and including the highest dose tested (1000 mg/kg bw/day)
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal body weights (both sexes and combined) noted by treatment up to 1000 mg/kg bw/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 1000 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal anogenital distance (both sexes) noted after treatment up to 1000 mg/kg bw/day.
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment with the
test item up to 1000 mg/kg bw/day.
One fetus with an external malformation was observed. The lower jaw of the affected fetus (in the high dose group) could not be seen, although small mandibles appeared present at skeletal examination. In addition, the cervical column of this fetus was malformed and during evisceration situs inversus was noted. As all these malformations were present in one fetus, this was regarded as incidental and not related to the test item.
External variations were not observed in any group.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment with the
test item up to 1000 mg/kg bw/day.
Skeletal malformations occurred in two fetuses at the low dose, besides the ones present in the multiple malformed high dose fetus described above. As these malformations (bent forelimb bones and supernumerary lumbar vertebra) occurred singly and in low dose fetuses they were considered chance findings. All skeletal variations occurred in the absence of a dose-related incidence trend and/or infrequently. Therefore, they were considered not related to treatment with the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment with the
test item up to 1000 mg/kg bw/day. Besides situs inversus observed in the grossly malformed high dose fetus described above, one fetus each at 300 and 1000 mg/kg bw/day had a small lens which was discovered at serial sectioning of heads. Due to the single occurrence these were considered not test item-related.
Visceral variations were observed for the liver (supernumerary lobe) and ureter (convoluted). These findings occurred infrequently and did not suggest any association with treatment with
the test item at any dose.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed up to and including the highest dose level tested (1000 mg/kg bw/day)
Abnormalities:
no effects observed
Developmental effects observed:
no

Results Dose Range Finding Study


No mortality occurred during the study period. No test item-related clinical signs were observed. Mean body weight (gain (corrected for gravid uterine weights)) and food consumption of treated females were unaffected by treatment with the test item. Macroscopic observations at necropsy did not reveal any alterations. All females were found to be pregnant with viable fetuses. One female had a early delivery. Number of corpora lutea, implantations, fetuses, live and dead fetuses, early and late resorptions were unaffected by treatment with the test item.
Pre- and post-implantation loss and mean fetal weight were considered unaffected by treatment with the test item.
Litter sizes were within normal limits for all groups. The male:female ratios were comparable between control and treated groups. External examination of the fetuses did not reveal any abnormalities.


Dose Formulation Analyses


The concentrations analyzed in the formulations of the low, mid and high dose groups were in agreement with target concentrations (i.e. mean accuracies between 94% and 105%). No test item was detected in the control group formulations. The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤ 5.0%)

Conclusions:
Based on the results of a Prenatal Developmental Toxicity Study in rats performed according to OECD/EC guidelines and GLP principles it is concluded that the NOAEL (maternal and developmental) for FDCA is 1000 mg/kg bw/day.
Executive summary:

A Prenatal Developmental Toxicity Study was performed in rats according to OECD/EC guidelines and GLP principles with FDCA via oral gavage at 100, 300 and 1000 mg/kg bw/day. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. No maternal toxicity and no developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. Based on the results of this study it is concluded that the NOAEL (maternal and developmental) for FDCA is 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A reliable study is available (Klimisch 1).
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

A Prenatal Developmental Toxicity Study was performed in rats according to OECD/EC guidelines and GLP principles with FDCA via oral gavage at 100, 300 and 1000 mg/kg bw/day. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation
loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations. 
Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.
No maternal toxicity and no developmental toxicity was observed in the 100, 300 and 1000 mg/kg/day groups. Based on the results of this study it is concluded that the NOAEL (maternal and developmental) for FDCA is >=1000 mg/kg bw/day.

Justification for classification or non-classification

Due to the absence of adverse systemic effects in the available toxicity studies on FDCA up to and including 1000 mg/kg bw/day, FDCA is not classified for reproductive and developmental toxicity.

Additional information