Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 1996 - 9 January 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted under GLP conditions and according to a guideline equivalent or similar to OECD guideline 416.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 113-138 g; Females: 101-128 g
- Housing: Individually under standard conditions
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum, municipal tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Batches of dosing formulation were prepared weekly by mixing amounts of Fyrolflex RDP appropriate to each dose level with powdered rodent chow.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Meal
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity under the conditions of this study was confirmed.
Duration of treatment / exposure:
(P) Males: 10 weeks prior to mating, during and up to 2-3 weeks of mating and until sacrifice
(P) Females: 10 weeks prior to mating, during and up to 2-3 weeks of mating, during gestation up to weaning of F1 (postnatal day 25) and until sacrifice
(F1) Males: 11 weeks starting at weaning, during and up to 2 weeks of mating and until sacrifice
(F1) Females: 10 weeks prior to mating, during and up to 2-3 weeks of mating, during gestation up to weaning of F1 (postnatal day 25) and until sacrifice
(F2) No treatment
Frequency of treatment:
Continuous (in diet)
Details on study schedule:
- Selection of parents from F1 generation when pups were 25 days (weaning) of age.
- Age at mating of the mated animals in the study: 14 weeks
Remarks:
Doses / Concentrations:
0, 1000, 10000, 20000 mg/kg (ppm) food
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 49, 520, 995 mg/kg bw/day
Basis:
analytical conc.
(P) males
Remarks:
Doses / Concentrations:
0, 59, 602, 1199 mg/kg bw/day
Basis:
analytical conc.
(P) females
Remarks:
Doses / Concentrations:
0, 55, 602, 1260 mg/kg bw/day
Basis:
analytical conc.
(F1) males
Remarks:
Doses / Concentrations:
0, 63, 683, 1411 mg/kg bw/day
Basis:
analytical conc.
(F1) females
No. of animals per sex per dose:
(P) 30 males and 30 females
(F1) 60 animals
Control animals:
yes, plain diet
Details on study design:
No data
Positive control:
Not relevant
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: morbidity and mortality, also parturition in dams during gestation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly prior to mating, but not during mating. Thereafter, males weekly until sacrifice, females on day 0, 6, 12, 18 and 20 of gestation and on postnatal day 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly prior to mating, but not during mating. After mating, male food consumption was measured weekly until sacrifice, female food consumption on day 0, 6, 12, 18 and 20 of gestation, on postnatal day 0, 4, 7, 14 and 21 and weekly thereafter.
Oestrous cyclicity (parental animals):
Daily vaginal smears were collected for the (P) and (F1) females for three weeks prior to mating to ensure and evaluate cyclicity. In addition, vaginal smears were collected and evaluated from these rats for two to three days prior to, and on the day of necropsy to determine the stage of the estrus cycle at termination
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals between end of mating and weaning of pups
- Maternal animals: All surviving animals after weaning of pups

GROSS NECROPSY
- Gross necropsy included examination of the external surface and pleural, peritoneal and cranial activities

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared and examined for gross lesions:
Vagina, uterus, ovaries with oviducts, cervix, stomach, pituitary, testes, epididymides, prostate, brain, liver, kidneys, adrenals, spleen, thymus, seminal vesicles.

Tissues below were also weighed:
Uterus, ovaries with oviducts, testes, epididymides, prostate, brain, liver, kidneys, adrenals, spleen, thymus, seminal vesicles.

Tissues below were also processed for histopathology:
Vagina, uterus, ovaries with oviducts, cervix, testes, epididymides, prostate, seminal vesicles.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 25 days and 21 days of age, respectively.
- These animals were not subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy of F1 included examination of the external surface and pleural, peritoneal and cranial activities. F2 generation rats were euthanized and discarded without necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
The F1 tissues indicated below were prepared weighed and examined for gross lesions, the tissues with an asterisk (*) were also processed for histopathology.
Vagina (*)
Uterus (*)
Ovaries with oviducts (*)
Cervix (*)
Stomach
Pituitary
Testes (*)
Epididymides (*)
Prostate (*)
Brain
Liver
Statistics:
ANOVA, followed by Dunnett's test were appropriate for body weights, body weight gains, food consumption and organ weight data.
Comparison of litter parameters was complicated by the fact that pup weight is dependent upon, among other factors, litter size. Litter size was accounted for by least squares comparisons for covariance.
Reproductive indices:
Not relevant
Offspring viability indices:
Not relevant
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One animal died without clinical signs that were considered treatment related

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Male food consumption and absolute body weight were significantly decreased during first week of test substance administration in high dose males. Only on postnatal day 21 food consumption was significantly decreased in females of the mid and high dose groups. These effects were most likely associated with flavor aversion to the test substance in the food and in dams also with concomitant decreased food consumption by the weanlings.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant increases observed in liver weight in male and female rats in a dose-related fashion, but these were attributed to metabolic adapation after histopathology. Increased adrenal weight was presumed to be related to stress associated with the initital flavor aversion and the resulting food avoidance in the mid and high dose groups.
Dose descriptor:
NOAEL
Effect level:
995 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No treatment related effects
Dose descriptor:
NOAEL
Effect level:
1 199 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No treatment related effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)
F1: Two animals died without clinical signs that were considered treatment related

BODY WEIGHT AND FOOD CONSUMPTION (OFFSPRING)
F1: Decreased body weight observed on postnatal day 7 and 14 until weaning, but this was considered related to the taste aversion phenomenon (decreased food consumption). Mean body weights decreased (significant for mid and high dose group) during 11-week pre-mating period, induced by decreased food consumption in the first 2 weeks.
F2: Decrease in body weight observed up on to postnatal day 21, which was considered to be related to litter size
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 260 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No treatment related effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 411 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No treatment related effects
Reproductive effects observed:
not specified

RESULTS OF TEST DOSING GROUPS
Control (plain diet) Low (1000 ppm) Medium (10000 ppm) High (20000 ppm)
ANALYSES
Actual concentration in diet Within 3% of target levels
Stability Confirmed
Homogeneity Confirmed
Test substance intake 0 mg/kg bw/day (P) M/F: 49 and 59 mg/kg bw/day (P) M/F: 520 and 602 mg/kg bw/day (P) M/F: 995 and 1199 mg/kg bw/day
0 mg/kg bw/day (F1) M/F: 55 and 63 mg/kg bw/day (F1) M/F: 602 and 683 mg/kg bw/day (F1) M/F: 1260 and 1411 mg/kg bw/day
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL
PARENTAL DATA (P)
Number of animals (M/F) 30/30 30/30 30/30 30/30
Mortality (numbers) x x x 1 female: dead during lactation
Body weight x x x Males: absolute weight significantly decreased in first week of treatment
Food consumption x x Females: significantly decreased on postnatal day 21 Males: significantly decreased in first week of treatment, Females: significantly decreased on postnatal day 21
Clinical signs x x x x
Organ weights x M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight
Macroscopy x x x x
Microscopy Hepatic periportal hypertrophy (adaptive) Not done Not done Hepatic periportal hypertrophy (adaptive)
Sperm count x x x x
Sperm motility x x x x
Sperm morphology x x x x
Length of oestrus cycle x x x x
Ovarian primordial follicle counts 82.4 +/- 33.8 Not done Not done 73.0 +/- 29.8
Number of females successful mated 29 27 28 29
Mating index (%) 97 90 93 97
Number selected for littering 27 25 28 29
No. of litters 23 25 28 26
Gestation index (%) 85 100 100 90
Litter size per dam (mean +/- SD) 12.3 +/- 1.80 11.7 +/- 2.72 11.7 +/- 2.51 11.0 +/- 2.6
PARENTAL DATA (F1)
Number of animals (M/F) 25/25 25/25 28/28 26/26
Mortality (numbers)
Body weight x M/F: Sporadic but significant differences M/F: Significant decrease throughout study. M: mean body weight gain significantly decreased M/F: Significant decrease throughout study. M: mean body weight gain significantly decreased
Food consumption M/F: Sporadic but significant differences x x
Clinical signs x x x x
Organ weights M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight
Macroscopy x x x x
Microscopy Hepatic periportal hypertrophy (adaptive) Not done Not done Hepatic periportal hypertrophy (adaptive)
Sperm count x x x x
Sperm motility x x x x
Sperm morphology x x x x
Length of oestrus cycle x x x x
Ovarian primordial follicle counts 73.4 +/- 18.9 Not done Not done 82.1 +/- 24.8
Number of females successful mated 25 25 26 26
Mating index (%) 100 100 93 100
No. of litters 23 25 22 24
Gestation index (%) 92 100 85 92
Litter size per dam (mean +/- SD) 12.1 +/- 3.45 12.9 +/- 3.02 12.7 +/- 1.73 11.6 +/- 2.67
 
OFFSPRING TOXICITY (F1)
Litter size (mean +/- SD) 12.3 +/- 1.80 11.7 +/- 2.72 11.7 +/- 2.51 11.0 +/- 2.6
Foetal weight (mean +/- SD) 6.78 +/- 0.327 6.84 +/- 0.428 6.89 +/- 0.528 7.05 +/- 0.466
Viability index (%) 97 99 94 96
Post natal survival until weaning (%, based on mean litter survival) 64 68 66 69
Visible abnormalities x x x x
Postnatal growth, growth rate (body weight gain) x x M/F: significant decrease PD14-21 F: significant decrease PD7-21, M: significant decrease PD14-21
Vaginal opening (F) or preputial separation (M) x x Delayed development (related to body weight) Delayed development (related to body weight)
OFFSPRING TOXICITY (F2)
Litter size (mean +/- SD) 12.1 +/- 3.45 12.9 +/- 3.02 12.7 +/- 1.73 11.6 +/- 2.67
Foetal weight (mean +/- SD) 6.84 +/- 0.501 6.69 +/- 0.448 6.73 +/- 0.410 6.87 +/- 0.385
Viability index (%) 98 98 96 97
Post natal survival until weaning (%, based on mean litter survival) 64 60 61 65
Postnatal growth, growth rate (body weight gain) x M/F: significantly decreased on PD4-21 M/F: significantly decreased on PD4-21 M/F: significantly decreased on PD4-21
x = no effects (as compared to control group)
Conclusions:
Under the conditions of this two-generation reprotoxicity study, Fyrolflex RDP induced no adverse effects in doses up to 20,000 ppm in parental, F1 and F2 animals. Therefore, a NOAEL of 20,000 ppm was established - representing measured concentrations of 995, 1199, 1260 and 1411 mg/kg bw/day for P: males, P: females, F1: males, F1: females, respectively. The substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.
Executive summary:

In this two-generation reproduction toxicity study, male and female rats (P and F1) were exposed to 0, 1000, 10000 or 20000 ppm Fyrolflex RDP in diet for approximately 11 weeks prior to mating, during and up to 2 -3 weeks of mating and until sacrifice (males) or during gestation up to the weaning of the pups (females). Mortality, clinical signs, body weights, food consumption and organ weights were recorded, reproductive function was assessed and gross necropsy and histopathology were performed.

The administered concentrations were analytically determined to be 0, 49, 520 and 995 mg/kg bw/day for males and 0, 59, 602 and 1199 mg/kg bw/day for females (P). For F1, this was 0, 55, 602 and 1260 mg/kg bw/day for males and 0, 63, 683 and 1411 mg/kg bw/day for females.

No treatment related adverse effects were observed in P, F1 or F2 (no necropsy performed). Effects that were seen were related to flavor aversion to the test substance in food, resulting in a decrease in food consumption and alterations in body and organ weights. Liver effects could be attributed to a metabolic adaptation to the test substance.

Under the conditions of this two-generation reprotoxicity study, Fyrolflex RDP induced no adverse effects in doses up to 20,000 ppm in parental, F1 and F2 animals. Therefore, a NOAEL of 20,000 ppm was established - representing measured concentrations of 995, 1199, 1260 and 1411 mg/kg bw/day for P: males, P: females, F1: males, F1: females, respectively. The substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
995 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Study duration:
subacute
Species:
rat
Additional information

In a two-generation reproduction toxicity study, male and female rats (P and F1) were exposed to 0, 1000, 10000 or 20000 ppm RDP in the diet for approximately 11 weeks prior to mating, during and up to 2 -3 weeks of mating and until sacrifice (males) or during gestation up to the weaning of the pups (females). Mortality, clinical signs, body weights, food consumption and organ weights were recorded, reproductive function was assessed and gross necropsy and histopathology were performed.

The administered concentrations were analytically determined to be 0, 49, 520 and 995 mg/kg bw/day for males and 0, 59, 602 and 1199 mg/kg bw/day for females (P). For F1, this was 0, 55, 602 and 1260 mg/kg bw/day for males and 0, 63, 683 and 1411 mg/kg bw/day for females.

No treatment related adverse effects were observed in P, F1 or F2 (no necropsy performed). Effects that were seen were related to flavor aversion to the test substance in food, resulting in a decrease in food consumption and alterations in body and organ weights. Liver effects could be attributed to a metabolic adaptation to the test substance. Under the conditions of this two-generation reprotoxicity study, RDP induced no adverse effects in doses up to 20,000 ppm in parental, F1 and F2 animals. Therefore, a NOAEL of 20,000 ppm was established - representing measured concentrations of 995, 1199, 1260 and 1411 mg/kg bw/day for P: males, P: females, F1: males, F1: females, respectively.

 


Short description of key information:
Two-generation oral (diet) reproductive toxicity study (OECD Guideline 416), rats: not reprotoxic

Effects on developmental toxicity

Description of key information

Two Developmental toxicity studies are available (rat, rabbit). Both studies were conducted following the OECD 414 test guidelines and according to requirements of GLP principles.

1.      Whitehead 2019: Fyrolflex RDP: Study for Effects on Embryo-Fetal Development in the Sprague-Dawley Rat by Oral Gavage Administration including Measurement of Plasma, Erythrocytes and Brain Cholinesterase Activity.

The purpose of this study was to assess the influence of Fyrolflex RDP, an industrial chemical, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat and to assess blood and brain cholinesterase activity in the pregnant rat and fetuses.

Three groups of 20 females received Fyrolflex RDP at doses of 40, 200 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group (Group 1) received the vehicle, corn oil, at the same volume dose (5 ml/kg/day) as the treated groups. Satellite animals (ten per group) were added for the purpose of cholinesterase assessment and a second Control group (Group 2) was added to produce additional control data for cholinesterase activity in the absence of historical control data for pregnant females. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

There were no deaths and no adverse clinical signs were observed following dose administration. Dosing signs were limited to salivation in all treated groups and chin rubbing at 200 and 1000 mg/kg/day, which was considered to be associated with palatability of the formulated test material.

There was no adverse effect of treatment on body weight or gravid uterus weight. There were no macro-pathology findings that were attributable to treatment.

Mean brain cholinesterase was not shown to be inhibited in pregnant females. There was no conclusive effect of treatment on erythrocyte cholinesterase activity, as a statistically significant difference (p<0.05) was identified between Control Group 2 and Control Group 1 for mean erythrocyte cholinesterase in pregnant females. Statistically significant dosagerelated mean plasma cholinesterase inhibition was evident in pregnant females at 40, 200 and 1000 mg/kg/day when compared to both Control Groups.

Mean brain cholinesterase and erythrocyte cholinesterase was not inhibited in fetuses. There was no conclusive effect of treatment on plasma cholinesterase activity in fetuses.

There were no adverse effects on litter parameters (implantations, pre- and post-implantation loss, live litter size and sex ratio), placental weights, total litter weights or overall fetal weights at any dose level and fetal development was not compromised at doses up to and including 1000 mg/kg/day.

Based on these results it was concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival, growth and development was 1000 mg/kg/day. The NOEL for brain and erythrocyte inhibition in pregnant females, and brain, erythrocyte and plasma cholinesterase inhibition in fetuses was 1000 mg/kg/day. However, the no observed effect level (NOEL) for inhibition of plasma cholinesterase activity in pregnant females was not established and is below the lowest dose investigated 40 mg/kg/day

2.      Ryan2009: A Developmental toxicity study of orally administered Fyroflex RDP in rabbits.

Pregnant rabbits were exposed to RDP by oral gavage from gestation day 6 through 29 in a concentration of 0, 50, 200 and 1000 mg/kg bw/day. No clinical signs of maternal toxicity were evident during the study or were apparent from gross necropsy observations. Mean body weights, body weight gains, food consumption, uterus weights and organ weights showed no test substance related effects. No significant differences in fetal (litter) body weights were observed between the test substance-treated groups and the vehicle control. Also, no significant increases in fetal deaths, resorptions, or malformations were observed between the test substance-treated groups and the control. The visceral, cephalic and skeletal malformations observed were low in incidence, sporadic in nature, and were not considered test substance-related because no clear pattern or relationship between the affected structure and dose group could be established. All anomalies were similar to or below the historical control values for anomalies in litter as published by the Middle Atlantic Reproduction and Teratology Association/Midwest Teratology Association (MARTA/MTA).

Under the conditions of the study, exposure to RDP by oral gavage up to 1000 mg/kg bw/day was found not to result in maternal toxicity or fetus developmental toxicity in rats

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 1995 - October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed under GLP conditions and according to a protocol equivalent or similar to OECD guideline 414.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
treatment period according to old guideline (exposure GD 6-29 in stead of complete gestation period)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hazleton Research Products (HRP), Inc., Denver, PA
- Age at study initiation: approx. 4.5 months (females)
- Weight at study initiation: 2.92-3.44 kg (females)
- Housing: under standard conditions
- Diet (e.g. ad libitum): Ad libitum, 50:50 mixture of high fiber and rabbit chow
- Water (e.g. ad libitum): Ad libitum, municipal water
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25
- Humidity (%): 30-69
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Doses of the test substance at 50, 200 and 1000 mg/kg bw/day were prepared in a corn oil vehicle at a constant dosing volume of 1.5 ml/kg bw. Individual doses were prepared daily, mixing the contents using a stopcock connector.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not necessary, accepted vehicle
- Amount of vehicle (if gavage): 1.5 ml/kg
- Lot/batch no. (if required): 114H0275
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: not indicated
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- Any other deviations from standard protocol:
Duration of treatment / exposure:
23 days (gestation days 6 through 29)
Frequency of treatment:
Once daily
Duration of test:
29 days
Remarks:
Doses / Concentrations:
50, 200, 1000 mg Fyrolflex RDP/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
31.5, 125.8, 629 mg RDP/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
27
Control animals:
yes, concurrent vehicle
Details on study design:
Not relevant
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice (weekdays) or once (weekends) daily
- Cage side observations: mortality

DETAILED CLINICAL OBSERVATIONS: Yes, clinical examinations
- Time schedule: Prior to randomization and study initiation

BODY WEIGHT: Yes
- Time schedule for examinations: at randomization, every two days during gestation, at study termination

FOOD CONSUMPTION:
- Food consumption recorded: Yes, but not corrected for spillage
- Time schedule: Day 0-29 (every 2 days) and at termination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 (followed by gross necropsy)
- Organs examined: Liver, spleen, kidneys (histopathology)
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 resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: one-third per litter
Statistics:
- Mean and standard deviation (all parameters)
- Multivariate analysis of variance
- One-way ANOVA
- Post-hoc comparisons: Dunnett's test (in presence of significant main effects)
- Chi-square and Fischer Exact test (malformation data)
Indices:
Not relevant
Historical control data:
Historical control values for anomalies in litter as published by the Middle Atlantic Reproduction and Teratology Association/Midwest Teratology Association (MARTA/MTA) were used.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- No mortality related to treatment
- No dose-related clinical signs observed in any of the substance-treated groups
- No significant differences detected in body weight, uterus weight, corrected body weight or corrected body weight gain
- No biologically significant effect on food consumption
- No significant effects observed on mean liver, kidney and spleen weight
- No gross pathological abnormalities noted
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No maternal toxicity up to the highest dose tested
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- No statistically significant difference in number of live or total implants, resorptions, corpora lutea, or percent implantation loss
- No significant difference found in mean fetal weight
- No gross external malformations observed in the pups
- No dose related increase in incidence nor any clear pattern of visceral anomalies was noted. The fetal incidence is similar to or below the observed incidence for these anomalies as published by MARTA/MTA
- The incidence of cephalic anomalies was greatest in the high dose group, but is similar or well below the observed incidence for these anomalies as published by MARTA/MTA
- No skeletal anomalies were considered related to treatment. No skeletal anomalies were observed in the high dose group. The fetal incidence is similar to or below the observed incidence for these anomalies as published by MARTA/MTA
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects were not observed up to the highest dose tested.
Key result
Abnormalities:
no effects observed
Developmental effects observed:
not specified

RESULTS OF TEST DOSING GROUPS
Control (vehicle) Low (50 mg/kg bw/day) Medium (200 mg/kg bw/day) High (1000 mg/kg bw/day)
MATERNAL TOXIC EFFECTS BY DOSE LEVEL 
Number of animals 27 27 27 27
Mortality and day of death 0 0 0 0
Body weight x x x Day 16-18: Significant increase in body weight gain
Food consumption x x x Day 20: Significant increase in food consumption
Clinical signs x x x x
Number pregnant per dose level 26 27 25 26
Number aborting 0 0 0 0
Number of corpora lutea 238 231 241 254
Number of implantations 234 227 226 236
Number of resorptions early/late 14/3 5/7 9/2 7/10
Pre-implantation loss 4 4 15 18
Post-implantation loss 17 12 11 17
Duration of Pregnancy x 2 animals: Premature delivery 1 animal: Parturition on GD 29 x
Number of litters 25 25 25 26
Gross pathology incidence and severity 1 animal: mass on the left Fallopian tube, 1 animal: non-viable litter observed x x x
Organ weight changes (liver, kidney, spleen) x x x x
FETAL DATA 
Litter size (mean +/- SD) 8.35 +/- 2.23 8.60 +/- 1.91 8.60 +/- 1.85 8.42 +/- 1.70
Number viable 217 215 215 219
Litter weights x x x x
Postnatal survival x x x Higher incidence of deaths in one litter
Sex ratio (M/F) 1:1 0.9:1 0.9:1 1.3:1
Grossly external abnormalities 1 pup: abnormal hindlimb x x x
Visceral abnormalities 1 pup: short/absent innominate, 1 pup: abnormal branching of the subclavian 1 pup: common truncus, 1 pup: short/absent innominate, 2 pup of 1 litter: abnormal branching of the subclavian x 1 pup: short/absent innominate
Skeletal abnormalities 1 pup: fused ribs 1 fetus: fused sternebrae, 2 pups: fused or forked ribs 3 fetuses: fused sternebrae, 1 pup: unilateral absence of lumbar vertebral arch x
Cephalic abnormalities x 1 pup: slight hypoplasia of diencephalon 1 pup: unilateral dilatation of ventricle 3 pups of 2 litters: unilateral dilatation of ventricle, slight hypoplasia of diencephalon or ectopic lens and convoluted retina
x = no effects (as compared to control group)
Conclusions:
Under the conditions of this study, exposure to Fyrolflex RDP by oral gavage up to 1000 mg/kg bw/day was found not to result in maternal and developmental toxicity in rabbits. Therefore, a NOAEL of 1000 mg/kg bw/day was established. As a result, the substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.
Executive summary:

Pregnant rabbits were exposed to Fyrolflex RDP by oral gavage from gestation day 6 through 29 in a concentration of 0, 50, 200 and 1000 mg/kg bw/day. Throughout the gestation period the dams were observed for clinical signs and body weight, food consumption was recorded. On day 29 of gestation the dams were sacrificed and post-mortem the liver, spleen and kidneys were weighed and examined. Uteri and ovaria were examined: gravid uterus weight and number of corpora lutea, implantations and resorptions was recorded. Litter was examined for gross external, visceral, cephalic and skeletal anomalies.

No clinical signs of maternal toxicity were evident during the study or were apparent from gross necropsy observations. Mean body weights, body weight gains, food consumption, uterus weights and organ weights showed no test substance relaeted effects.

No significant differences in fetal (litter) body weights were observed between the test substance-treated groups and the vehicle control. Also, no significant increases in fetal deaths, resorptions, or malformations were observed between the test substance-treated groups and the control. The visceral, cephalic and skeletal malformations observed were low in incidence, sporadic in nature, and were not considered test substanc-related because no clear pattern or relationship between the affected structure and dose group could be established. All anomalies were similar to or below the historical control values for anomalies in litter as published by the Middle Atlantic Reproduction and Teratology Association/Midwest Teratology Association (MARTA/MTA).

Under the conditions of this study, exposure to Fyrolflex RDP by oral gavage up to 1000 mg/kg bw/day was found not to result in maternal and developmental toxicity in rats. Therefore, a NOAEL of 1000 mg/kg bw/day was established. As a result, the substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Per ECHA decision number CCH-D-2114360752-49-01/F (attached)
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018 (current)
Deviations:
yes
Remarks:
addition of measurement of CHE in brain plasma and erythorocyte in pregnant females and fetuses
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998 (Current)
Deviations:
yes
Remarks:
addition of measurement of CHE in brain plasma and erythorocyte in pregnant females and fetuses
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau
Version / remarks:
November 24, 2000 (current)
Deviations:
yes
Remarks:
addition of measurement of CHE in brain plasma and erythorocyte in pregnant females and fetuses
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
Pursuant to Article 41(1) and (3) of the REACH Regulation, ECHA has requested this pre-natal developmental toxicity study (test method: EU B.31./OECD TG 414) in a second species (rat) by the oral route with inclusion of measurement of plasma, erythrocyte and brain cholinesterase activity.
Deviations:
yes
Remarks:
addition of measurement of CHE in brain plasma and erythorocyte in pregnant females and fetuses
Principles of method if other than guideline:
Additional measurements were reqested by ECHA (described in the attached document). These meaurements included investigation of brain cholinesterase activity, as well as plasma and erythorocytes cholinesterase activity.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Intended use: Industrial chemical.
Appearance: Clear pale yellow viscous liquid.
Storage conditions: At ambient temperature (10 to 30C).
Supplier: Sponsor.
Batch number: 17 185 K19
Expiry date: 21 November 2019
Purity: Considered to be 100%.

COA attached
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Strain/Species: Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Duration of acclimatization before commencement of pairing: 5 days for the main phase.
12 days for the satellite phase.
Age of the animals at the start of the study (Day 0 of gestation): 68 to 81 days old for the main phase
75 to 88 days old for the satellite phase
Weight range of the animals at the start of the study (Day 0 of gestation): 216 to 278 g
Route of administration:
oral: gavage
Details on exposure:
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Individual dose volume: was calculated from the most recently recorded scheduled body weight.
Frequency: Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Item Preparation and Analysis
Formulation:
Group Treatment Dose Nominal concentration Formulated concentration Volume dose
(mg/kg/day) (mg/ml) (mg/mL) (mL/kg)

1 Vehicle 0 0 0 5
2 Vehicle 0 0 0 5
3 Fyrolflex RDP 40 8 8 5
4 Fyrolflex RDP 200 40 40 5
Fyrolflex RDP 1000 200 200 5

Correction factor: None.
Vehicle: Corn oil.
Method of preparation: The required amount of test item was weighed. Approximately 40% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order.

Frequency of preparation Weekly. Formulations were prepared in advance of the first day of dosing.
Storage of formulation Refrigerated (2 to 8C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity Specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Envigo Study
Number: RD67DH). In that study, formulations in the concentration range 1 mg/mL to 200 mg/mL were confirmed to be stable for:
One day stored at ambient temperature (15 to 25C).
15 days stored refrigerated (2 to 8C).
Achieved concentration Samples of each formulation prepared for administration on Day 6 after mating for the main phase and Day 19 after mating for the satellite phase were analyzed for achieved concentration of the test item.
Analysis The report of method of analysis and results is attached.
Details on mating procedure:
3.3.2 Mating
Main phase
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Satellite phase
Wet smears Before pairing, using pipette lavage.
Male/female ratio and female criteria for pairing 1:1 with identified stock males.
Only females expected to mate (i.e. in proestrus), were paired with stock males; pairing on the day of proestrus controlled the mating’s.
No more than ten females were mated on each day.
Male/female separation Morning after pairing.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
3.3.3 Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method Main phase - to group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
Satellite phase - allocations were non-sequential.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.
Duration of treatment / exposure:
From Day 6 to 19 after mating.
Frequency of treatment:
Once daily
Duration of test:
From Day 6 to 19 after mating.
Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Dose / conc.:
40 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
200 mg/kg bw/day
Remarks:
mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
Dose / conc.:
0 other: ml/kg/day
Remarks:
Vehicle Control Corn Oil
Dose / conc.:
0 other: ml/kg/day
Remarks:
A second Control group was added to produce additional control data for cholinesterase activity in the absence of historical control data for pregnant females.
No. of animals per sex per dose:
20 females received Fyrolflex RDP per treatment group and corn oil control group.
Satellite animals included ten females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
Three groups of 20 females received Fyrolflex RDP at doses of 40, 200 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating.
A similarly constituted Control group (Group 1) received the vehicle, corn oil, at the same volume dose (5 ml/kg/day) as the treated groups.
Satellite animals (ten per group) were added for the purpose of cholinesterase assessment and a second Control group (Group 2) was added to produce additional control data for cholinesterase activity in the absence of historical control data for pregnant females.
Animals were killed on Day 20 after mating for reproductive assessment and 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. All satellite phase females the brain weight was recorded. All main phase fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.
Ovaries and uterine content:
The following were recorded for all animals:
Uterus- Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn
Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).3.7.4 Fetal Examination and Processing - Main Phase

Fetal examinations:
Fetal Examination
Examination of all viable fetuses and placentae. Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.
Statistics:
The following data types were analyzed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Blood cholinesterase (plasma and erythrocyte)
Brain cholinesterase
Organ weights, absolute
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 3, 4 and 5
Group 2 vs 3, 4 and 5
Group 1 vs 2
Additinal details are attached.
Historical control data:
Available (attached)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse clinical signs were observed following dose administration. Dosing signs were limited to salivation in all treated groups and chin rubbing at 200 and 1000 mg/kg/day, which was considered to be associated with palatability of the formulated test material and not indicators of toxic effect.
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect of treatment on body weight. Overall bodyweight gains from Day 6-20 were marginally increased (8 to 10%) at all dose levels when compared to both Control groups. Mean gravid uterine weight was increased by 10-11% at 1000 mg/kg/day compared to Control Group 2 and Control Group 1 (p<0.05), however, adjusted bodyweight change from Day 6-20 was unaffected by treatment. (Fig 1 attachment).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was statistically increased at 1000 mg/kg/day over Days 10-14 of gestation when compared to both Control groups (Control Group 1: p<0.05, Control Group 2: p<0.01) and at 200 and 1000 mg/kg/day over Days 18-20 of gestation when compared to Control Group 1 (p<0.05 and p<0.01, respectively). These differences were, however, only 1 or 2 g higher than the mean value for both control groups and were considered not toxicologically significant.
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
Description (incidence and severity):
The reduction in mean absolute brain weight observed at 1000 mg/kg/day when compared to Control Group 2 (93.1%, p<0.05), was considered not toxicologically significant as it did not differ significantly from the Control Group 1 mean.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Description (incidence and severity):
Mean brain cholinesterase was not shown to be inhibited in pregnant females. There was no conclusive effect of treatment on erythrocyte cholinesterase activity, as a statistically significant difference (p<0.05) was identified between Control Group 2 and Control Group 1 for mean erythrocyte cholinesterase in pregnant females. Statistically significant dosage related mean plasma cholinesterase inhibition was evident in pregnant females at 40, 200 and 1000 mg/kg/day when compared to both Control Groups.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Results
The mean formulated concentrations were within 9% of the nominal concentration, confirming the accuracy of formulation. The difference from mean values remained within 2%, confirming precise analysis. Procedural recovery values ranged between 92.3% and 107.3% of the fortified concentration, confirming the continued accuracy of the analytical method.
There were no deaths and no adverse clinical signs were observed following dose administration. Dosing signs were limited to salivation in all treated groups and chin rubbing at 200 and 1000 mg/kg/day, which was considered to be associated with palatability of the formulated test material.
There was no adverse effect of treatment on body weight or gravid uterus weight. Overall bodyweight gains from Day 6-20 were marginally increased at all dose levels when compared to both Control groups. Gravid uterine weights were statistically significantly increased at 1000 mg/kg/day compared to Control Group 1 (p<0.05) but not Control Group 2, however, adjusted bodyweight change from Day 6-20 was unaffected by treatment.
Mean food consumption was statistically increased at 1000 mg/kg/day over Days 10-14 of gestation when compared to both Control groups (Control Group 1: p<0.05, Control Group 2: p<0.01) and at 200 and 1000 mg/kg/day over Days 18-20 of gestation when compared to Control Group 1 (p<0.05 and p<0.01, respectively). These differences were, however, only 1 or 2 g higher than the mean value for both control groups and were considered not toxicologically significant.
Mean brain cholinesterase was not shown to be inhibited in pregnant females. There was no conclusive effect of treatment on erythrocyte cholinesterase activity, as a statistically significant difference (p<0.05) was identified between Control Group 2 and Control Group 1 for mean erythrocyte cholinesterase in pregnant females. Statistically significant dosage related mean plasma cholinesterase inhibition was evident in pregnant females at 40, 200 and 1000 mg/kg/day when compared to both Control Groups.
Mean brain cholinesterase and erythrocyte cholinesterase was not inhibited in fetuses. There was no conclusive effect of treatment on plasma cholinesterase activity in fetuses.
There were no macropathology findings that were attributable to treatment.
There were no adverse effects on litter parameters (implantations, pre- and post-implantation loss, live litter size and sex ratio), placental weights, total litter weights or overall fetal weights at any dose level and fetal development was not compromised at doses up to and including 1000 mg/kg/day.
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
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Oral administration of Fyrolflex RDP to Sprague Dawley rats during Days 6-19 of gestation at doses of 40, 200 and 1000 mg/kg/day was well tolerated and did not result in any deaths or change in the clinical condition of the pregnant females.

All females that mated were pregnant. The following assessment is based on up to 30 females per group with live young at termination on Day 20 of gestation.
There was no marked or conclusive effect of treatment upon litter parameters (corpora lutea, implantations, resorptions, implantation loss, live young or sex ratio).

The no effect level for brain and erythrocyte cholinesterase activity in pregnant females was 1000 mg/kg/day. A no effect level for plasma cholinesterase activity could not be established. These findings did not produce any adverse effect on the clinical condition, bodyweights, food consumption, brain weight or macropathology of the adult females.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No effects of maternal toxicity were observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental weights, total litter weights and overall fetal weights were unaffected by treatment
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
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants were consistent with historical control data and show no relationship to treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants were consistent with historical control data and show no relationship to treatment.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no adverse effect on litter parameters (corpora lutea, implantations, resorptions, implantation loss, live young or sex ratio), placental weights, total litter weights or overall fetal weights at any dose level and fetal development was not compromised at doses up to and including 1000 mg/kg/day.
In fetuses, the no effect level for mean brain, erythrocyte and plasma cholinesterase was 1000 mg/kg/day
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects found
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Conclusions:
Based on these results it was concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival, growth and development was 1000 mg/kg/day. The NOEL for brain and erythrocyte inhibition in pregnant females, and brain, erythrocyte and plasma cholinesterase inhibition in fetuses was 1000 mg/kg/day. However, the no observed effect level (NOEL) for inhibition of plasma cholinesterase activity in pregnant females was not established and is below the lowest dose investigated, 40 mg/kg/day.
Executive summary:

Summary

The purpose of this study was to assess the influence of Fyrolflex RDP, an industrial chemical, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague-Dawley rat and to assess blood and brain cholinesterase activity in the pregnant rat and fetuses.

Three groups of 20 females received Fyrolflex RDP at doses of 40, 200 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group (Group 1) received the vehicle, corn oil, at the same volume dose (5 ml/kg/day) as the treated groups. Satellite animals (ten per group) were added for the purpose of cholinesterase assessment and a second Control group (Group 2) was added to produce additional control data for cholinesterase activity in the absence of historical control data for pregnant females.

Animals were killed on Day 20 after mating for reproductive assessment and 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 satellite phase females the brain weight was recorded. All main phase fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

The mean formulated concentrations were within 9% of the nominal concentration, confirming the accuracy of formulation. The difference from mean values remained within 2%, confirming precise analysis. Procedural recovery values ranged between 92.3% and 107.3% of the fortified concentration, confirming the continued accuracy of the analytical method.

There were no deaths and no adverse clinical signs were observed following dose administration. Dosing signs were limited to salivation in all treated groups and chin rubbing at 200 and 1000 mg/kg/day, which was considered to be associated with palatability of the formulated test material.

There was no adverse effect of treatment on body weight or gravid uterus weight. Overall bodyweight gains from Day 6-20 were marginally increased at all dose levels when compared to both Control groups. Gravid uterine weights were statistically significantly increased at 1000 mg/kg/day compared to Control Group 1 (p<0.05) but not Control Group 2, however, adjusted bodyweight change from Day 6-20 was unaffected by treatment.

Mean food consumption was statistically increased at 1000 mg/kg/day over Days 10-14 of gestation when compared to both Control groups (Control Group 1:p<0.05, Control Group 2: p<0.01) and at 200 and 1000 mg/kg/day over Days 18-20 of gestation when compared to Control Group 1 (p<0.05 andp<0.01, respectively). These differences were, however, only 1 or 2 g higher than the mean value for both control groups and were considered not toxicologically significant.

Mean brain cholinesterase was not shown to be inhibited in pregnant females. There was no conclusive effect of treatment on erythrocyte cholinesterase activity, as a statistically significant difference (p<0.05) was identified between Control Group 2 and Control Group 1 for mean erythrocyte cholinesterase in pregnant females. Statistically significant dosage‑related mean plasma cholinesterase inhibition was evident in pregnant females at 40, 200 and 1000 mg/kg/day when compared to both Control Groups.

Mean brain cholinesterase and erythrocyte cholinesterase was not inhibited in fetuses. There was no conclusive effect of treatment on plasma cholinesterase activity in fetuses.

There were no macropathology findings that were attributable to treatment.

There were no adverse effects on litter parameters (implantations, pre- and post-implantation loss, live litter size and sex ratio), placental weights, total litter weights or overall fetal weights at any dose level and fetal development was not compromised at doses up to and including 1000 mg/kg/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:
subchronic
Experimental exposure time per week (hours/week):
7
Species:
other: rats and rabbits
Quality of whole database:
klimisch score 1- studies was conducted under OECD 414 guidleines and according to GLP principles.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the result that both the NOAEL for reproduction toxicity and developmental toxicity exceed the NOAELs for maternal toxicity, reproduction and developmental toxicity is not expected. Therefore, it is concluded that RDP does not need to be classified as toxic to reproduction or development according to the criteria laid down in Annex VI of the EEC Council Directive 67/548/EEC (amended by Directive 83/467/EEC), and outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).

Additional information