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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A study on the Reproduction toxicity is required by REACH for dossier of FR-513. At this point we request to waive this investigation. For the following justification: A 28- day oral repeat dose toxicity study in the rat with a 14 day recovery period was conducted including investigation of some important reproduction endpoints (completed 2015). The results showed no systemic toxicity effects and the No Observed Adverse Effect level (NOAEL) was determined as >500 mg/kg/day (highest dose tested). No treatment related changes in sperm count and motility were observed (data are attached in Table 7 and Appendix 8). Vaginal lavages which were taken early morning during the 3 week period from all females, prior to termination of the animals showed no treatment related changes in the oestrus cycle (data are attached as Appendix 8, in 7. X of the IUCLID file). In addition, there were no dose related changes in organ weight of ovaries, seminal vesicles, testis, ureter, uterus, vagina in comparison to control animals. From the generated scientific data it can be concluded that subacute repeated exposure to FR-513, does not seem to induce adverse effect on the fertility nor cause systemic toxicity or affect the reproductive organs. In addition, an OECD 414 Developmental toxicity study was conductedfor FR-513 without any adverse findings on developmental toxicity or maternal toxicity. The results are summarised in section 7.8.2. Based on the lack of adverse effect to the reproduction endpoints that were investigated during the 28 day study and the results of the developmental study ICL requests a waiver from performing further reproduction studies.

Effects on developmental toxicity

Description of key information

Study for Effects on Embryo-Fetal Development in Sprague-Dawley Rats by Oral Gavage Administration was conducted following OECD 414 and according to GLP stabdards.

Treatment of pregnant female Sprague Dawley rats with FR-513 at 1000 mg/kg/day from Day 6 of gestation was not tolerated with marked post dosing signs including underactive/unresponsive behaviour, partially closed eyelid(s), unsteady muscle reaction and prostrate posture following one to three doses. In two females these findings were so severe as to require termination after 2 or 3 doses, on Day 7 or Day 8 of gestation. Consequent to the reduction of the high dose level to 500 mg/kg/day (after the third day of dosing) these signs were less marked/no longer apparent.,

In females receiving 100 or 300 mg/kg/day, administration of FR-513 during Days 6 to Day 19 after mating was well tolerated with no toxicity related changes in clinical condition and no treatment related macroscopic findings at necropsy.

Treatment at 1000 / 500 mg/kg/day elicited mean body weight loss during Days 6-7 of gestation (after the first dose), and lower weight gain and food consumption during Days 6-9 of gestation, with lower gravid uterine weight and lower mean maternal weight gain following adjustment for the contribution of the gravid uterus. Three females at the high dose group that received only 500 mg/kg/day from the start of dosing (on Day 6 of gestation) showed similar but minimal maternal body weight loss (3-4 g) after a single dose, but mean gain for Days 6 to 9 was higher than controls, and food intake was only marginally lower during the same period. Effects on body weight and food consumption of this magnitude are considered not to be adverse.

Treatment at 100 or 300 mg/kg/day had no discernible effect upon gestating females.

Embryo-fetal survival, growth and development were considered to have been unaffected by treatment at 100, 300 or 1000 / 500 mg/kg/day.

Based on the results of this study in Sprague Dawley rats, it was concluded that, although the initial maternal response to treatment at 1000 mg/kg/day was severe, subsequent reaction to treatment at 500 mg/kg/day was considered to not adverse and this level can be considered the No Adverse Effect Level (NOAEL)..

Within the context of this study, the NOAEL for embryo-fetal survival, growth and development was also set at 500 mg/kg/day.

 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Certificate of Analysis is attached.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Environmental Control:

* Rodent facility- Limited access was allowed, to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
* Air supply - Filtered fresh air which was passed to atmosphere and not recirculated.
* Temperature and relative humidity - Monitored and maintained within the range of 20-24ºC and 40-70%.
There were no deviations from these ranges.

* Lighting - Artificial lighting, 12 hours light : 12 hours dark.
* Electricity supply - Public supply with automatic stand-by generators.

Animal Accommodation:
* Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent which was
changed daily during pairing.

*Cage distribution - The cages constituting each group were blocked by group and mounted in batteries.
Bedding. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

*Number of animals per cage:
Acclimatization - up to four animals
During pairing - one (stock) male and one female
Gestation - one female

*Diet Supply - SDS VRF1 Certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

*Water Supply - Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.


Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis
The homogeneity and stability of formulations during storage were determined as part of Envigo Study No. AFH0038. In that GLP compliant study, homogeneity and stability of FR-513 in corn oil formulations (at nominal concentrations of 6 and 100 mg/mL) was confirmed during magnetic stirring for up to two hours, and on re-suspension following storage at ambient temperature for one day and refrigeration for up to 10 days.
On this current study the homogeneity and stability of FR 513 in corn oil formulations at the nominal concentration of 200 mg/mL was confirmed during magnetic stirring for up to four hours, and on re-suspension following storage at ambient temperature (nominally 21°C) for up to 18 days and refrigeration (nominally 2 - 8°C) for up to 8 days.
However, after 8 days refrigerated storage, crystalline material was apparent that required heating and significant stirring to dissolve. Crystalline material was not observed following ambient storage. It was therefore recommended to store formulations at 200 mg/mL at ambient temperature.
Details on mating procedure:
Two groups of 20 females received FR-513 at doses of 100 or 300 mg/kg/day by oral gavage administration, once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg. A third treated group of 20 females commenced treatment with FR-513 at 1000 mg/kg/day, however, this dose level was subsequently reduced to 500 mg/kg/day shortly after commencement of treatment due to the severity of the post dosing signs observed. Due to the spread of mating dates, three females in this group received the dose of 500 mg/kg/day throughout the entire treatment period. All other females received at least one dose of 1000 mg/kg/day before the reduced dose level of 500 mg/kg/day was implemented. Females were dosed once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg.
A similarly constituted Control group received the vehicle, corn oil, at the same volume dose and for the same duration as the treated groups.
Animals were killed on Day 20 after mating for reproductive assessment and detailed fetal examination.
Duration of treatment / exposure:
19 consequtive days
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
Females were treated from Day 6 to Day 19 (inclusive) after mating.
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
A third treated group of 20 females commenced treatment with FR-513 at 1000 mg/kg/day, however, this dose level was subsequently reduced to 500 mg/kg/day shortly after commencement of treatment due to the severity of the post dosing signs observed. Due to the spread of mating dates, three females in this group received the dose of 500 mg/kg/day throughout the entire treatment period. All other females received at least one dose of 1000 mg/kg/day before the reduced dose level of 500 mg/kg/day was implemented.
No. of animals per sex per dose:
Two groups of 20 females received FR-513 at doses of 100 or 300 mg/kg/day by oral gavage administration, once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg. A third treated group of 20 females commenced treatment with FR-513 at 1000 mg/kg/day, however, this dose level was subsequently reduced to 500 mg/kg/day shortly after commencement of treatment due to the severity of the post dosing signs observed. Due to the spread of mating dates, three females in this group received the dose of 500 mg/kg/day throughout the entire treatment period. All other females received at least one dose of 1000 mg/kg/day before the reduced dose level of 500 mg/kg/day was implemented.
Control animals:
yes, concurrent vehicle
Details on study design:
Females were dosed once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg.
A similarly constituted Control group received the vehicle, corn oil, at the same volume dose and for the same duration as the treated groups.
Animals were killed on Day 20 after mating for reproductive assessment and detailed fetal examination.
.
Maternal examinations:
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded.


Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus- Gravid uterine weight (including cervix and ovaries).
The following was recorded for all animals (including those permanently sacrificed) : For each ovary/ uterine horn- number of: Corpora lutea, implantation sites, resorption sites (classified as early or late, fetuses (live and dead).
Fetal examinations:
All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.
Examination of all viable fetuses and placentae- Dissected from the uterus, individually weighed and identified within the litter and examined externally with sex of each fetus recorded.
50% of the fetuses in each litter were eviscerated, fixed and stained.
Statistics:
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) x 100
Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100
Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values.
Clinical signs:
no effects observed
Description (incidence and severity):
Treatment of pregnant female Sprague Dawley rats with FR-513 at 1000 mg/kg/day from Day 6 of gestation was not tolerated with marked post dosing signs including underactive/unresponsive behaviour, partially closed eyelid(s), unsteady muscle reaction and prostrate posture following one to three doses. In two females these findings were so severe as to require termination after 2 or 3 doses, on Day 7 or Day 8 of gestation. Consequent to the reduction of the high dose level to 500 mg/kg/day (after the third day of dosing) these signs were less marked/no longer apparent.,
In females receiving 100 or 300 mg/kg/day, administration of FR-513 during Days 6 to Day 19 after mating was well tolerated with no toxicity related changes in clinical condition and no treatment related macroscopic findings at necropsy.
Treatment at 1000 / 500 mg/kg/day elicited mean body weight loss during Days 6-7 of gestation (after the first dose), and lower weight gain and food consumption during Days 6-9 of gestation, with lower gravid uterine weight and lower mean maternal weight gain following adjustment for the contribution of the gravid uterus. Three females at the high dose group that received only 500 mg/kg/day from the start of dosing (on Day 6 of gestation) showed similar but minimal maternal body weight loss (3-4 g) after a single dose, but mean gain for Days 6 to 9 was higher than controls, and food intake was only marginally lower during the same period. Effects on body weight and food consumption of this magnitude are considered not to be adverse.
Treatment at 100 or 300 mg/kg/day had no discernible effect upon gestating females.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment of pregnant female Sprague Dawley rats with FR-513 at 1000 mg/kg/day from Day 6 of gestation was not tolerated In two females these findings were so severe as to require termination after 2 or 3 doses, on Day 7 or Day 8 of gestation. Consequent to the reduction of the high dose level to 500 mg/kg/day (after the third day of dosing) these signs were less marked/no longer apparent.,
In females receiving 100 or 300 mg/kg/day, administration of FR-513 during Days 6 to Day 19 after mating was well tolerated with no toxicity related changes in clinical condition and no treatment related macroscopic findings at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 / 500 mg/kg/day mean body weight loss (2%) was observed after the first dose, during Days 6-7 of gestation, attaining statistical significance compared with the Control value (no change). Mean body weight loss after the first dose for the three females that only received 500 mg/kg/day was of a slightly lower magnitude (3-4 g) to the mean loss observed for the remainder of the group ( 7g, range +5 to -16g) but values for individual animals were within a range comparable to that observed for control females over the same period (+13 to -15g
Mean body weight gain from day 6 to day 9 of gestation was lower in those females receiving at least one dose of 1000 mg/kg/day (6g) compared to those receiving only the 500 mg/kg/day dose over the same period (10g) and control mean gain (8g), with values for individual animals generally reflecting the number of doses at the higher level received. Thereafter body weight gain was generally similar to Controls and unaffected by treatment.
At 100 or 300 mg/kg/day body weight change during gestation was similar to Controls and unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption during Days 6-9 of gestation for females receiving 1000 / 500 mg/kg/day were slightly lower than Controls (4 g/day lower), attaining statistical significance. For the remainder of gestation mean values of food consumption were similar to Controls. Food consumption during Days 6-9 of gestation for the three females that only received 500 mg/kg/day was comparable with other females in the group (18-19 g).
Food consumption at 100 or 300 mg/kg/day was similar to Controls throughout gestation and unaffected by treatment.
Food efficiency:
not specified
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):
no effects observed
Description (incidence and severity):
Signs of chin rubbing and salivation were observed in animals of all treated groups, however, this was considered to relate to general distaste of the formulation rather than any effect of toxicity
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean gravid uterine weight at 1000 / 500 mg/kg/day was marginally lower than Controls (6 g
lighter), and when adjusted for the contribution of the gravid uterus, maternal body weight
gain was also slightly lower than Controls (22 g vs. 28 g in the Control group), attaining
statistical significance.
Mean gravid uterine weights and adjusted body weight gain at 100 or 300 mg/kg/day were
similar to Controls, and unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related findings at macroscopic examination of the adult females at any of the dose levels investigated.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no major fetal abnormalities that were considered related to treatment. One major abnormality was found in a fetus from the high dose group and there were two fetuses in two litters in the control group with major abnormalities of the jaw, one of which also had a cleft palate.At 1000 mg/kg/day there was a slightly increased incidence of the minor abnormalities medially thickened/kinked ribs; delayed/ incomplete ossification/ unossified cranial centres, hyoid, cervical vertebrae and pelvic bones; partially undescended thymus and brain haemorrhages compared to concurrent control, however, all parameters are within concurrent Historical Control Data (HCD) ranges (in terms of litter incidence) and are considered unrelated to treatment.
At 300 mg/kg/day there was a slightly increased incidence of the minor abnormalities delayed/ incomplete ossification/ unossified pelvic bones compared to concurrent control however, the incidence was within the concurrent Historical Control Data (HCD) range and was considered unrelated to treatment.
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
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Non were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no major fetal abnormalities that were considered related to treatment. One major abnormality was found in a fetus from the high dose group and there were two fetuses in two litters in the control group with major abnormalities of the jaw, one of which also had a cleft palate.
At 1000 mg/kg/day there was a slightly increased incidence of the minor abnormalities
medially thickened/kinked ribs; delayed/ incomplete ossification/ unossified cranial centres,
hyoid, cervical vertebrae and pelvic bones; partially undescended thymus and brain
haemorrhages compared to concurrent control, however, all parameters are within concurrent
Historical Control Data (HCD) ranges (in terms of litter incidence) and are considered
unrelated to treatment.
At 300 mg/kg/day there was a slightly increased incidence of the minor abnormalities
delayed/ incomplete ossification/ unossified pelvic bones compared to concurrent control
however, these incidences are within concurrent Historical Control Data (HCD) ranges and
are considered unrelated to treatment.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested for teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
no
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the results of this study in Sprague Dawley rats, it was concluded that, although the initial maternal response to treatment at 1000 mg/kg/day was severe, subsequent reaction to treatment at 500 mg/kg/day was considered to not adverse and this level can be considered the No Adverse Effect Level (NOAEL)..
Within the context of this study, the NOAEL for embryo-fetal survival, growth and development was also set at 500 mg/kg/day.
Executive summary:

Summary

The purpose of this study was toassess the influence of FR-513 (a flame retardant) on embryo-fetal survival and development in the Sprague-Dawley Rat when administered during the organogenesis and fetal growth phases of pregnancy.

Two groups of 20 females received FR-513 at doses of 100 or 300 mg/kg/day by oral gavage administration, once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg. 

A third treated group of 20 females commenced treatment with FR-513 at 1000 mg/kg/day, however, this dose level was subsequently reduced to 500 mg/kg/day shortly after commencement of treatment due to the severity of the post dosing signs observed. Due to the spread of mating dates, three females in this group received the dose of 500 mg/kg/day throughout the entire treatment period. All other females received at least one dose of 1000 mg/kg/day before the reduced dose level of 500 mg/kg/day was implemented. Females were dosed once daily from Day 6 to 19 after mating at a dose volume of 5 mL/kg.   

A similarly constituted Control group received the vehicle, corn oil, at the same volume dose and for the same duration as the treated groups. 

Animals were killed on Day 20 after mating for reproductive assessment and detailed fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

Treatment of pregnant female Sprague Dawley rats with FR-513 at 1000 mg/kg/day from Day 6 of gestation was not tolerated, with two females killed for welfare reasons after showing marked signs including underactive/unresponsive behaviour, partially closed eyelid(s), unsteady muscle reaction and prostrate posture following one to three doses.

After lowering this dose to 500 mg/kg/day, and for females receiving 100 or 300 mg/kg/day, administration of FR-513 during Days 6 to Day 19 after mating was well tolerated by pregnant female rats, with no further deaths and no toxicity related changes in the clinical condition of the adult females

Signs of chin rubbing and salivation were observed in animals of all treated groups, however, this was considered to relate to general distaste of the formulation rather than any effect of toxicity.

At 1000 / 500 mg/kg/day mean body weight loss during Days 6-7 of gestation (after the first dose), and low food consumption during Days 6-9 of gestation were observed. Mean gravid uterine weight and body weight change when adjusted for the contribution of the gravid uterus were also slightly lower than Controls at this dose level.

At 100 or 300 mg/kg/day body weight change during gestation, body weight change when adjusted for the gravid uterine weight, and food consumption were similar to Controls and unaffected by treatment.

There were no treatment related findings at macroscopic examination of the adult females at any of the dose levels investigated.

Embryo-fetal survival, growth and development were considered to have been unaffected by treatment at 100, 300 or 1000 / 500 mg/kg/day.

Conclusion

Based on the results of this study in Sprague Dawley rats, it was concluded that, although the initial maternal response to treatment at 1000 mg/kg/day was severe, subsequent reaction to treatment at 500 mg/kg/day was considered to not adverse and this level can be considered the No Adverse Effect Level (NOAEL).

Within the context of this study, the NOAEL for embryo-fetal survival, growth and development was also set at 500 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K-1

Justification for classification or non-classification

Study for Effects on Embryo-Fetal Development in Sprague-Dawley Rats by Oral Gavage Administration was conducted following OECD 414 and according to GLP stabdards.Based on the results of this study of effects upon embryo-fetal developmental in Sprague Dawley rats, it was concluded that the No-Adverse-Effect-Level (NOAEL) for FR-513 upon maternal toxicity was 500 mg/kg/day, as no maternal reaction to treatment occurred that was considered to be adverse. Within the context of this study, the NOAEL for embryo-fetal survival, growth and development was also set at 500 mg/kg/day. Therefore, classification is not justified.