Registration Dossier

Administrative data

Description of key information

Fyrolflex RDP: Txicity study by oral gavage administration to CD rats for 13 weeks including measurements of plasma, erythrocyte and brain Cholinesterase Activity (2019).

28 days repeated dose inhalation toxicity study in rats (Johnson 1994).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998
Deviations:
yes
Remarks:
includes measurements of brain, plasma and erythrocytes CHE per ECHA request
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Test item identity (including alternative names): Fyrolflex RDP, Tetraphenyl m-phenylene bis(phosphate), Phosphoric acid, 1,3-phenylene tetraphenyl ester
EC number 260-830-6
CAS number 57583-54-7
Intended use Industrial chemical -flame retardant.
Appearance: Clear pale yellow viscous liquid.
Storage conditions: At ambient temperature (10 to 30°C).
Batch number 17 185 K19
Expiry date 21 November 2019
Purity Considered to be 100%.
COA attached
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Information
Age of the main study animals at start of treatment: 55 to 61 days.
Weight range of the main study animals at the start of treatment: Males: 270 to 378 g
Females: 186 to 260 g
Allocation Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed 20% of the mean for the appropriate sex. No replacements were necessary.

Animal Care and Husbandry
Environmental Control
Animal facilityLimited access - 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%.
On two occasions 30 April 2018 and 1 May 2018, the temperature was recorded at 19°C. Although conditions were occasionally outside the indicated ranges, these deviations were minor and of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
3.4.2 Animal Accommodation
Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution Males and females were blocked by group and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage Up to four of the same sex.
Bedding Wood based bedding which was changed at appropriate intervals each week.
3.4.3 Environmental Enrichment
Aspen chew block Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.
3.4.4 Diet Supply
Diet Teklad 2014C Diet.
Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry).
3.4.5 Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.
Route of administration:
oral: gavage
Details on route of administration:
Administration Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth., at constant doses in mg/kg.
Volume dose: 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1 and 2) Vehicle at the same volume dose as the treated groups.
Frequency: Once daily at approximately the same time each day.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, linearity of detector response, repeatability and method accuracy and precision.
The homogeneity and stability was confirmed for Fyrolflex RDP in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for up to 1 day and refrigerated storage for up to 15 days.
The mean concentrations of Fyrolflex RDP in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation with the exception of Week 1 Group 4 that was -20.0%. The mean concentrations of Fyrolflex RDP are presented in Table 4 of Annex 2 (attached).
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Fyrolflex RDP
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Fyrolflex RDP
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Fyrolflex RDP
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Fyrolflex RDP
No. of animals per sex per dose:
Four groups (Groups 3, 4, 5 and 6), each comprising 10 male and 10 female Sprague Dawley rats, received Fyrolflex RDP at doses of 30, 100, 300 or 1000 mg/kg/day.
A similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups.
A further 10 male and 10 female satellite animals were allocated to Group 1, a control group and Groups 3, 4 and 6.
A further 5 male and 5 female satellite animals were allocated to Group 5.
An additional 10 male and 10 female Sprague-Dawley rats were assigned to Group 2 as satellite animals as an additional control group. They were used for blood cholinesterase (plasma and erythrocyte) and brain cholinesterase evaluations.
Control animals:
yes
Details on study design:
Rationale for Dose Level Selection
Dose levels of 30, 100, 300 and 1000 mg/kg/day were selected following completion of the preliminary 14-day Rangefinding (RF) study Envigo Study No. XD56VG (attached).In the RF study there were no effects considered as adverse on clinical condition, body weights, food consumption, blood chemistry or macropathology. Liver weights in the preliminary study were high at all dose levels and in both sexes, but as the enzymes indicative of liver damage were unaffected,
this was considered not sufficiently adverse to decrease the high dose for this study.
Inhibition of plasma cholinesterase was also noted in both sexes, however in the absence of any changes in cholinesterase activity in the brain and erythrocytesafter two weeks of treatment, the finding was also considered not adverse.

An additional vehicle Control group was incorporated to provide additional Control data for all cholinesterase assessments (in the absence of recent historical control data for this age range)
A fourth dose group was included to explore the broadest dose range, with the intention of identifying a NOAEL. Therefore, the high dose level for this study is
1000 mg/kg/day with the two intermediate and low dose levels chosen to facilitate the determination of a dose response.
Observations and examinations performed and frequency:
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Clinical Signs
A detailed weekly physical examination was performed on each animal to monitor general health.

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment.

The following measurements, reflexes and responses were recorded:
Approach response
Pinna reflex
Auditory startle reflex
Tail pinch response
Grip strength
Motor Activity
Sacrifice and pathology:
All main study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule Main study animals were killed following 13 weeks of treatment.
Sequence To allow satisfactory inter-group comparison.
Other examinations:
Body Weight: The weight of each animal was recorded before treatment commenced (Day -5 for Groups 1, 3-6 only), on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.
Food Consumption: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded before treatment started and for each week throughout the study.
Water Consumption: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.
Ophthalmic Examination: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope (at the discretion of the examining veterinary surgeon a slit-lamp biomicroscope may also be used).
Hematology, Peripheral Blood: Blood samples were collected after overnight withdrawal of food and prior to dosing
Blood Chemistry: Blood samples were collected after overnight withdrawal of food and prior to dosing.
Cholinesterase Analyses
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs associated with treatment. Clinical signs recorded in all groups are commonly occurring findings and the incidence and distribution were unrelated to treatment.
Signs associated with dosing included chin rubbing in 9/10 males and 6/10 females at 30 mg/kg/day and in all males and females at 100, 300 and 1000 mg/kg/day. This finding was also evident in 3/10 and 2/10 males in Control groups 1 and 2, respectively.
Salivation was evident in 4/10 males and 7/10 females at 100 mg/kg/day. At 300 and 1000 mg/kg/day, salivation was observed in all males and females.
These signs are considered to be reactions to the palatability of the test item, and not indicators of toxic effect.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A statistical significant increase (114.9%, p<0.05) was identified between Control Group 1 and 2 for overall mean bodyweight change over Weeks 1-13 in males. This resulted in significantly low overall mean bodyweight gains over Weeks 13 at all treatment levels when compared to Control Group 2 (p<0.01) but since there was no significant difference from Control Group 1, the results for all but the high dose group were considered inconclusive. Only at 1000 mg/kg/day was overall mean bodyweight change over 13 Weeks significantly low when compared to both Control Group 1 (83.7%, p<0.05) and Control Group 2 (72.9%, p<0.01).
Over Weeks 1-4 of study, mean bodyweight gain in males at all treatment levels was slightly low when compared to Control Group 1 and significantly low (p<0.05 at 30, 100 and 300 mg/kg/day: p<0.01 at 1000 mg/kg/day) when compared to the slightly higher gains observed in Control Group 2. Over Weeks 4-8, mean bodyweight gain in males at 300 mg/kg/day was significantly low when compared to Control Group 2 (p<0.01) and at 1000 mg/kg/day was statistically significantly low when compared to Control Groups 1 and 2 (p<0.01).
Overall mean body weight gain for females at all dose levels was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Overall mean food consumption over Weeks 1-13 identified a dose-dependent increase when compared to Control Group 1 (Males: 103.3, 104.7, 110.0 and 114.0%; Females: 103.9, 106.9, 109.8 and 117.6%) at 30, 100, 300 and 1000 mg/kg/day, respectively. Although when compared to Control Group 2, only the food intake of males receiving 1000 mg/kg/day or females receiving 300 or 1000 mg/kg/day were slightly higher in value.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment related ophthalmic findings. Any findings in Week 13 were observed prior to treatment, with the exception of one finding. The faint opacity of the cornea (right eye) of one male receiving 1000 mg/kg/day was an isolated, commonly occurring finding and, therefore, was considered due to biological variation.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haematological examination of peripheral blood performed after 13 weeks of treatment revealed statistically significant low mean cell haemoglobin at 300 and 1000 mg/kg/day in males and at 100, 300 and 1000 mg/kg/day in females (males: 94.3% (p<0.01), 93.8% (p<0.01); females: 97.0% (p<0.05), 95.4% (p<0.01) and 92.9% (p<0.01)), respectively, when compared with Control Group 1 and mean cell hemoglobin concentration at 300 and 1000 mg/kg/day in males and at 30, 100, 300 and 1000 mg/kg/day in females (males: 95.1% (p<0.01), 95.1% (p<0.01); females: 98.6% (p<0.05), 98.3% (p<0.05), 97.2% (p<0.01) and 96.0% (p<0.01), respectively, when compared with Control Group 1.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were some minor changes to clinical pathology parameters however the degree of change was minimal. The low alkaline phosphatase concentrations was indicative of an effect on liver function, however this was considered to be adaptive due to the type of histopathological changes seen.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity (as measured by approach response, pinna reflex, auditory startle reflex, tail pinch response) and grip strength were unaffected by treatment with Fyrolflex RDP. As presented in Table 3, Appendix 3 of the full report.
Motor activity was unaffected by treatment with Fyrolflex RDP as indicated in Figure 1, Table 4, Appendix 4 of the attached full report.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean adjusted liver weights showed a statistically significant dose-dependent increase in males and females at 30, 100, 300 and 1000 mg/kg/day (Males: 109.4% (p<0.05), 112.3% (p<0.01), 118.3% (p<0.01) and 130.0% (p<0.01); Females: 114.6% (p<0.01), 130.4% (p<0.01), 155.6% (p<0.01) and 155.6% (p<0.01)), respectively when compared with Control.
The mean adjusted adrenal weights showed a dose-dependent increase in females at 30, 100, 300 and 1000 mg/kg/day (103.5%, 115.8% (p<0.05), 117.5% (p<0.05) and 119.3% (p<0.05)), respectively when compared with Control, although no similar finding was evident in males.
All other organs were unaffected by treatment. (Table 10, Appendix 11 of the full report).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Abnormally dark livers were seen in two females receiving 300 mg/kg/day and seven females receiving 1000 mg/kg/day.
The incidence and distribution of all other findings were considered to be unrelated to treatment.

There were some minor changes to clinical pathology parameters however the degree of change was minimal. The low alkaline phosphatase concentrations was indicative of an effect on liver function, however this was considered to be adaptive due to the type of histopathological changes seen.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight hepatocyte hypertrophy was seen in the liver of males and females given 100, 300 or 1000 mg/kg/day which correlated with high liver weights observed in both sexes and dark livers observed in 2 females receiving 300 mg/kg/day and 7 females receiving 1000 mg/kg/day. Diffuse hepatocyte hypertrophy of the liver is often due to an increase in activity of the hepatic microsomal drug metabolizing enzymes as a consequence of induced microsomal metabolism by the test substance and is considered an adaptive change (Greaves 2011).
Minimal or slight follicular cell hypertrophy was seen in the thyroids of almost all males and females given 100, 300 or 1000 mg/kg/day. Hypertrophy/hyperplasia of the follicular epithelium in the thyroids is considered a secondary phenomenon due to altered clearance of thyroid hormones as a consequence of increased microsomal enzyme activity by the liver (Greaves 2011). This results in an increase in thyroid stimulating hormone (TSH), causing follicular cell stimulation. This is supported by good correlation between liver and thyroid findings in individual animals of both sexes.
Histopathological findings: neoplastic:
no effects observed
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 adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg/day. There was no adverse effect of treatment at any dose level, the detailed behavioral assessments were unaffected and there were no adverse histopathological findings.
Remarks on result:
other:
Remarks:
No effects observed that were treatment related
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
other: No effects observed
Organ:
other: no adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg/day. There was no adverse effect of treatment at any dose level, the detailed behavioral assessments were unaffected and there were no adverse histopathological findings.
Conclusions:
Based on these results it was concluded that the no adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg/day. There was no adverse effect of treatment at any dose
level, the detailed behavioral assessments were unaffected and there were no adverse histopathological findings.
Executive summary:

SUMMARY

The purpose of this study was to assess the systemic toxic potential, including assessment of plasma, erythrocyte and brain cholinesterase activity, of Fyrolflex RDP when administered to CD rats by oral gavage over a 13-week period.

Four groups (Groups 3, 4, 5 and 6), each comprising ten male and ten female Sprague-Dawley rats, received Fyrolflex RDP at doses of 30, 100, 300 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups. A further ten male and ten female satellite animals were allocated to Group 1, a control group and Groups 3, 4 and 6. A further five male and five female satellite animals were allocated to Group 5. An additional ten male and ten female Sprague-Dawley rats were assigned to Group 2 as satellite animals as an additional control group. They were used for blood cholinesterase (plasma and erythrocyte) and brain cholinesterase evaluations.

During the study, clinical observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, blood and brain cholinesterase activity, organ weight,

macropathology and histopathology investigations were undertaken.

Results

The homogeneity and stability was confirmed for Fyrolflex RDP in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for up to 1 day and refrigerated storage for up to 15 days. The mean concentrations of Fyrolflex RDP in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation with the exception of Week 1 Group 4 that was -20.0%.

There were no deaths and no treatment related clinical signs were observed following dose administration. Dosing signs were limited to salivation at 100, 300 and 1000 mg/kg/day and chin rubbing in all treated groups, considered to be associated with palatability of the

formulated test material. There was no effect of treatment on sensory reactivity, grip strength, motor activity or

ophthalmic examination. The haematological examination of peripheral blood performed after 13 weeks of treatment revealed statistically significant low mean cell haemoglobin at 300 and 1000 mg/kg/day in males and at 100, 300 and 1000 mg/kg/day in females (males: 94.3% (p<0.01), 93.8%

(p<0.01); females: 97.0% (p<0.05), 95.4% (p<0.01) and 92.9% (p<0.01)), respectively, when compared with Control and mean cell hemoglobin concentration at 300 and 1000 mg/kg/day in males and at 30, 100, 300 and 1000 mg/kg/day in females (males: 95.1% (p<0.01), 95.1%

(p<0.01); females: 98.6% (p<0.05), 98.3% (p<0.05), 97.2% (p<0.01) and 96.0% (p<0.01), respectively, when compared with Control.

Report Envigo Study Number: RD67DH Page 9.

The biochemical examination of the blood plasma performed during Week 13 of treatment for males and females revealed low or statistically significantly low alkaline phosphatase concentrations at 30, 100, 300 and 1000 mg/kg/day (males: 87.3%, 73.5% (p<0.05), 88.2% (p<0.05), 71.6% (p<0.01); females: 51.3% (p<0.01), 46.3% (p<0.01), 38.8% (p<0.01) and 40.0% (p<0.01)), respectively, when compared with Control. Total protein values were statistically significantly increased in males and females at 30, 100, 300 and 1000 mg/kg/day

(males: 104.6% (p<0.05), 104.6% (p<0.05), 104.6% (p<0.05), 106.2 (p<0.01); females: 101.4%, 107.0% (p<0.05), 108.5% (p<0.01) and 114.1% (p<0.01)), respectively when compared with Control.

Statistically significant, dosage-related differences in mean plasma cholinesterase was evident in males and females at 30, 100, 300 and 1000 mg/kg/day when compared to both Control Groups. Mean erythrocyte cholinesterase was statistically significantly inhibited in

females at 1000 mg/kg/day when compared to Control Group 1 and 2 (71.8% p<0.05 and 65.1% p<0.01, respectively). At 1000 mg/kg/day, mean erythrocyte cholinesterase in males was also low when compared to Control Groups 1 and 2, however this was not statistically significant (79.8%

and 81.5%, respectively). Mean brain cholinesterase in males at 1000 mg/kg/day was statistically significantly low when compared to Control Group 2 (95.2% p<0.05), however there value was comparable to the Control Group 1 mean and no similar finding was evident in

females.

Mean adjusted liver weights showed a statistically significant dose-dependent increase in males and females at 30, 100, 300 and 1000 mg/kg/day (Males: 109.4% (p<0.05), 112.3% (p<0.01), 118.3% (p<0.01) and 130.0% (p<0.01); Females: 114.6% (p<0.01), 130.4%

(p<0.01), 155.6% (p<0.01) and 155.6% (p<0.01)), respectively when compared with Control.

The mean adjusted adrenal weights showed a dose-dependent increase in females at 30, 100, 300 and 1000 mg/kg/day (103.5%, 115.8% (p<0.05), 117.5% (p<0.05) and 119.3% (p<0.05)), respectively when compared with Control, although no similar finding was evident in males.

Abnormally dark livers were seen in two females receiving 300 mg/kg/day and seven females receiving 1000 mg/kg/day. Minimal or slight hypertrophy of the hepatocytes was seen in males and females dosed at 100, 300 or 1000 mg/kg/day and minimal or slight hypertrophy of the follicular cells was seen in males and females dosed at 100, 300 or 1000 mg/kg/day.

Conclusion

Based on these results it was concluded that the no adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg/day. There was no adverse effect of treatment at any dose level, the detailed behavioral assessments were unaffected and there were no adverse histopathological findings.

Endpoint conclusion
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:
rat
Quality of whole database:
Reliable K-1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 December 2003 - 7 March 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 412 and under GLP conditions.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
in addition, MNSE activity was determined to study neurotoxicity
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: Charles River Laboratories
- Age at study initiation: 8-9 weeks
- Weight at study initiation:
Males: 196-233 g
Females: 167-212 g
- Housing: According to guideline
- Diet (e.g. ad libitum): Ad libitum, rodent chow
- Water (e.g. ad libitum): Ad libitum, tap water
- Acclimation period: 3 days (of 15 days quaratine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-26
- Humidity (%): 32-60
- Air changes (per hr): 15 +/- 2
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: By Quartz Crystal Microbalance (QCM)
MMAD: 1.39-1.70 um
GSD: 1.67-2.04 um
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 52-port nose-only inhalation exposure chambers
- Method of holding animals in test chamber: Holding tubes
- Source and rate of air: Regulated compressed air source
- System of generating particulates/aerosols: nebulizers and regulated compressed air source
- Air flow rate: Adjusted to generate aerosol with particle size of <3 um MMAD
- Treatment of exhaust air: Moved through an inertial trap and a particulate filter by a ring compressor and exhausted outside the building

TEST ATMOSPHERE
- Brief description of analytical method used:
Aerosol mass concentraiton: Gravimetric analysis (filter), light scattering method (sensors) and chemical analysis (filter)
Aerosol particle size: Quartz Crystal Microbalance (QCM)
- Samples taken from breathing zone: no, only from filters and sensors
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetry and chemical analysis
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day 5 days/week
Remarks:
Doses / Concentrations:
0.1, 0.5, 2.0 mg/l
Basis:
nominal conc.
No. of animals per sex per dose:
Low- and mid-dose group: 10
High-dose group: 20
Control animals:
other: filtered air
Details on study design:
- Rationale for animal assignment: Random (constrained by body weight)
- Post-exposure recovery period in satellite groups: 60 days
- Section schedule rationale: Random
Positive control:
Not relevant
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 7 days prior to exposure
- Anaesthetic used for blood collection: Yes (CO2 or sodium pentobarbital at termination)
- Animals fasted: Yes (overnight before termination)
- How many animals: All
- Parameters checked:
APTT
Erythrocyte count and morphology
Hematocrit
Hemoglobin concentration
Leukocyte count (total and differential)
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Mean corpuscular volume
Platelet count
Prothrombin time
Reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (overnight before termination)
- How many animals: All
- Parameters checked:
Albumin
Albumin/globulin ratio (calculated)
Bilirubin (total)
Blood urea nitrogen
Calcium
Chloride
CPK
Cholesterol
Cholinesterase (plasma)
Cholinesterase (erythrocyte)
Creatinine
Gamma glutamyl transpeptidase
Globulin
Glucose
LDH
Monocyte non-specific esterase
Phosphorus
Potassium
Serum alanine aminotransferase
Serum alkaline phosphatase
Serum aspartase aminotransferase
Sodium
Total protein
Triglycerides
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, examination of external surface and all orifices; external surfaces of brain and spinal cord; organs and tissues of cranial, thoracic, abdominal and pelvic cavities and neck; remainder of the carcass

ORGAN WEIGHTS: Yes, see below:
Liver
Kidney
Lungs
Spleen
Adrenal glands
Heart
Brain
Testes with epididymides or ovaries
Pituitary
Thymus
Thyroid/parathyroids

At recovery only lung weight determined

HISTOPATHOLOGY: Yes, microscopic examination of tissue of control and high-dose group (+ tissues with lesions and target organs from low- and mid-dose group):
Adrenal glands
Brain
Buccal mucosa
Epididymides
Esophagus
Eyes
Heart
Kidneys
Larynx
Liver
Lungs
Nose
Ovaries
Pancreas
Pituitary gland
Spinal cord
Spleen
Testes
Thymus
Thyroid gland
Trachea

Gross lesions
Target organs
Other examinations:
Not relevant
Statistics:
ANOVA followed by Dunnett's test for comparison of multiple groups with a single control group
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN
Mean body weight significantly decreased in high-dose males in weeks 1-3, and did not recover during the first 5 weeks of recovery. No effect were found in females, exept for a significant increase in mean body weight for high-dose females in week 4 of recovery.

FOOD CONSUMPTION
Food consumption significantly decreased in high dose males during weeks 1 and 3 of exposure

HEMATOLOGY
Low-dose female rats had significantly increased total erythrocyte counts, hemoglobin and hematocrit.

CLINICAL CHEMISTRY
No toxicologically significant effects on any clinical chemistry parameter during exposure.

Mean plasma cholinesterase activity levels significantly decreased in high-dose males and mid- and high-dose females at end of exposure period. Significant decrease was still present in high-dose females at end of recovery. No effects on erythrocyte cholinesterase levels.

MNSE activity levels increased in low- and mid-dose rats at end of exposure period. In high-dose animals this activity was comparable to controls, not indicating a dose related effect.

ORGAN WEIGHTS
Dose-related increase in absolute and relative lung weights in test group, significantly in mid and high dose animals at end of exposure period. This persisted in high-dose animals during recovery.
Mean absolute liver weights significantly increased in high-dose females, and mean relative liver weight significantly increased in mid- and high-dose females and high-dose males after exposure period.

GROSS PATHOLOGY
All animals of high-dose group had confluent white foci in the lungs. This effect was still apparent after recovery and not seen in the control group animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Alveolar histiocytosis observed in all mid- and high-dose animals at end of exposure period. According to the athors, these changes are consistent with a response to foreign material (water insoluble liquid aerosol) and they were not considered to be a response to chronic injury. Chronic foreign body inflammation was observed in high-dose animals at end of recovery.
Dose descriptor:
NOEL
Effect level:
0.1 mg/L air (nominal)
Sex:
male/female
Basis for effect level:
other: Lung effects, decrease in plasma cholinesterase (females)
Critical effects observed:
not specified

Not relevant

Conclusions:
Under the conditions of this study, a No Observed Effect Level (NOEL) of 0.1 mg/L was established for the inhalation of Fyroflex RDP in rats. This NOEL was based on the presence of lung effects, which persisted after recovery.
Executive summary:

This study was performed to determine the ability of Fyroflex RDP to induce toxicity in rats by inhalation. The study was conducted according to OECD guideline 412 and under GLP conditions. Additionally, the Monocyte NonSpecific Esterase (MNSE) activity was evaluated to study neurotoxicity.

 

No deaths or toxicologically significant clinical signs were noted. In males of the high-dose group body weight and body weight gain was decreased. This returned to normal after recovery. Plasma cholinesterase activity was decreased in high-dose males and mid- and high-dose females, which persisted for high-dose females after recovery. No inhibition of erythrocyte cholinesterase acitivy levels was seen. MNSE activity levels were increased in low- and mid-dose rat, however, in high-dose animals this activity was comparable to that of controls. A dose-related increase in absolute and relative lung weights was seen in the test group, and was significant in mid- and high-dose animals. This persisted in high-dose animals during recovery. Mean absolute liver weights were significantly increased in high-dose females, and mean relative liver weight significantly increased in mid- and high-dose females and high-dose males. All animals of the high-dose group had confluent white foci in the lungs. This effect was still apparent after recovery and alveolar histiocytosis was observed in all mid- and high-dose animals. Chronic foreign body inflammation was observed in the high-dose animals at end of recovery.

Under the conditions of this study, a No Observed Effect Level (NOEL) of 0.1 mg/L was established for the inhalation of RDP in rats. This NOEL was based on the presence of lung effects, which persisted after recovery.

Endpoint conclusion
Dose descriptor:
NOAEC
100 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
90 -days Dermal toxicity study does not have to be conducted on RDP based on available experimental information and exposure route considerations.
2 Acute Dermal toxicity studies are available on RDP. In both studies no toxicity was observed (LD50 > 2000 mg/kg) and the substance did not induce any signs of systemic toxicity on the rats. In addition, RDP was not irritant to skin or eye, and did not cause allergic skin reactions in the Skin sensitisation study. Dermal absorption of RDP is limited as indicated by series of toxicokinetic studies.
The main route of exposure for RDP is the oral route. Nevertheless, the personal precautions for users indicate explicitly the obligation for using protective rubber gloves while handling the substance.
Therefore, for the reasons stated above and since the study is costly and requires use of many animals, we believe that performing this study is not necessary and can be avoided

Critical effects observed:
not specified

Additional information

A 28 days repeated dose nose-only inhalation study was performed with rats to determine the ability of test substance to induce toxicity in rats by inhalation. Rats were exposed in three dose groups (0.1, 0.5 and 2.0 mg/L) for 6 hrs per day, 5 days a week. The study was conducted according to OECD guideline 412 and under GLP conditions. Additionally, the Monocyte Non Specific Esterase (MNSE) activity was evaluated to study neurotoxicity. No deaths or toxicologically significant clinical signs were noted. In males of the high-dose group body weight and body weight gain was decreased. This returned to normal after recovery. Plasma cholinesterase activity was decreased in high-dose males and mid- and high-dose females, which persisted for high-dose females after recovery. No inhibition of erythrocyte cholinesterase activity levels was seen. MNSE activity levels were increased in low- and mid-dose rat, however, in high dose animals this activity was comparable to that of controls. A dose-related increase in absolute and relative lung weights was seen in the test group, and was significant in mid- and high-dose animals. This persisted in high-dose animals during recovery. Mean absolute liver weights were significantly increased in high-dose females, and mean relative liver weight significantly increased in mid- and high-dose females and high-dose males. All animals of the high-dose group had confluent white foci in the lungs. This effect was still apparent after recovery and alveolar histiocytosis was observed in all mid- and high-dose animals. Chronic foreign body inflammation was observed in the high-dose animals at end of recovery. Under the conditions of this study, a No Observed Effect Level (NOEL) of 0.1 mg/L was established for the inhalation of RDP in rats. This NOEL was based on the presence of lung effects, which persisted after recovery.

 

Two supporting studies are available, both repeated dose toxicity studies via intraperitoneal injection. In the first study, in which rats were exposed to RDP for a 28-day period, a NOAEL of 0.5 mg/kg bw/day was observed, based on decreased plasma cholinesterase, increased relative liver weight and histopathological effects. In the second study, rats were exposed to a dose of 500 mg/kg bw/day for a 14-day period and allowed a recovery period of 26 weeks, to determine reversibility of effects. The results indicated that when RDP is administered intraperitoneally to rats, it is slowly metabolised and removed from the body. Furthermore, it is indicated that the granulomatous inflammation seen histopathologically is caused by the slow metabolisation.


Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

Based on the results of the key study (90 days repeated dose oral toxicity in rats) it was concluded that the no adverse effect level (NOAEL) for systemic toxicity was 1000 mg/kg/day. There was no adverse effect of treatment at any dose level, the detailed behavioral assessments were unaffected and there were no adverse histopathological findings. Therefore, RDP does not need to be classified for repeated dose toxicity, in accordance withthe 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)

Based on the 28 days repeated dose inhalation study, RDP does not need to be classified for repeated dose toxicity. The effects seen at the lowest observed effect level are considered to be adaptive effects that were not observed after recovery. Based on the effects observed at the highest dose level, no classification is necessary in accordance withthe 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).

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