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Toxicological information

Neurotoxicity

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Administrative data

Description of key information

Key, CTL/AR7090; neurotoxicity (acute oral, single administration by gavage, rat, similar to OECD 424, GLP):

NOAEL (systemic toxicity): 25 mg/kg bw (males and females)

LOAEL (systemic toxicity): 60 mg/kg bw (males and females)

NOAEL (neurotoxicity): 60 mg/kg bw (females), ≥ 150 mg/kg bw (males)

LOAEL (neurotoxicity): 150 mg/kg bw (females)

 

Key, 91N0127; neurotoxicity (subchronic, repeated administration via diet, rat, similar to OECD 424, GLP):

NOAEL (systemic toxicity): 30 ppm (corresponding to 1.5 – 2.3 mg/kg bw/day for males and 1.8 – 2.6 mg/kg bw/day for females)

LOAEL (systemic toxicity): 125 ppm (corresponding to 5.6 – 10.2 mg/kg bw/day for males and 6.9 – 10.1 mg/kg bw/day for females)

NOAEL (neurotoxicity): 125 ppm (corresponding to 5.6 – 10.2 mg/kg bw/day for males and 6.9 – 10.1 mg/kg bw/day for females)

LOAEL (neurotoxicity): 500 ppm (corresponding to 22.8 – 40.2 mg/kg bw/day for males and 21.6 – 43.9 mg/kg bw/day for females)

 

Key, PFX 004/042634, neurotoxicity (developmental, repeated administration via diet, rat, EPA OPPTS 870.6300, GLP):

NOAEL (maternal systemic toxicity): 45 ppm (corresponding to 3.8 – 9.2 mg/kg bw/day)

LOAEL (maternal systemic toxicity): 90 ppm (corresponding to 6.6 – 18 mg/kg bw/day)

NOAEL (morphological development): 90 ppm (corresponding to 6.6 – 18 mg/kg bw/day)

LOAEL (morphological development): >90 ppm (corresponding to 6.6 – 18 mg/kg bw/day)

NOAEL (functional development): 20 ppm (corresponding to 1.6 – 4.1 mg/kg bw/day)

LOAEL (functional development): 45 ppm (corresponding to 3.8 – 9.2 mg/kg bw/day)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 Aug - 31 Aug 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
methodological limitations (See: Principles of methods)
Principles of method if other than guideline:
Deviations from OECD guideline 424:
No detailed clinical examination was performed within 8 hours after dosing;
Observations for mortality must be 2/day;
Positive control data were not reported;
Only gross pathology with determination of brain weights was performed at termination;
Histopathology was not performed on the nervous system.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Alpk:APfSD (Wistar derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Alderly Park, Macclesfield, Cheshire, UK
- Age at study initiation: at least 42 days
- Weight at study initiation: 144 - 190 g (males), 123 - 159 g (females)
- Fasting period before study: no
- Housing: 5 per cage, sexes separately
- Diet: CT1 diet (Special Diet Services Limited, Stepfield, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 Aug 2001 To: 31 Aug 2001
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose level, the appropriate amount of the test material was suspended in the vehicle. Each preparation was divided into 4 aliquots and a fresh aliquot was used for each day of dosing. The dosing formulations were stored at room temperature in the dark.

VEHICLE
- Concentration in vehicle: Nominal: 0, 1, 2.5, 6, 15 mg/mL
Analytical mean: 0, 0.95, 2.39, 5.9, 15.1 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance and test substance preparations in vehicle were confirmed by analysis. The stability ranged from 100 - 106.3% of the target concentration. The homogeneity of test substance preparations was confirmed by analysis and deviations within samples was up to 2.1%.
Duration of treatment / exposure:
Single exposure, postexposure period: 14 days
Frequency of treatment:
Single oral dose on Day 1 of the study
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
60 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
No. of animals per sex per dose:
10 per sex per group (+3 per sex as spares)
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily for mortality and clinical signs.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Pre-study (Week -1), on Day 1 (prior to dosing and 2.5 h after dosing) and on Days 8 and 15.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time scheudle: weekly intervals

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: No

LOCOMOTOR ACTIVITY: Yes
- Procedure: Locomotor activity was monitored by an automated activity recording apparatus, which records small and large movements as activity.
- Time schedule: pre-study (Week -1), Day 1 (approximately 2.5 h after treatment), and on Days 8 and 15
- Length of session, number and length of subsessions: each observation was divided into 10 scans of 5 min duration
- Total activity: not reported
- Ambulatory activity: not reported
- Large movements: yes
- Small movements: yes
Sacrifice and (histo)pathology:
- Time point of sacrifice: 15 days after administration of the test item, all animals were sacrificed by overexposure to halothane Ph. Eur. vapour followed by exsanguination.
- Brain weight: yes
- Length and width of brain: no
- Other: All gross pathological changes were examined.

No histopathology examination was performed.
Other examinations:
No
Positive control:
No
Clinical signs:
no effects observed
Description (incidence and severity):
There were no effects observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 10 and 25 mg/kg bw/day: No treatment-related effects were observed.
- 60 mg/kg bw/day: Body weights were statistically significantly lower than control values in both sexes on Day 8, and in females on Day 15. Mean body weights of male rats were 5% lower on Day 8. In females mean body weights were 4% and 6% lower on Day 8 and 15, respectively.
- 150 mg/kg bw/day: Statistically significant lower body weights in both sexes on Day 8 and 15 as compared to control animals. In males body weight was 8% and 6% lower on Day 8 and 15, respectively. In females reduction in body weight was about 5% on Day 8 and 15.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly reduced for males and females in the 60 and 150 mg/kg bw groups and for females in the 25, 60 and 150 mg/kg bw group during week 1. No effects on food consumption in males in the 10 and 25 mg/kg bw groups and in females in the 10 mg/kg bw group. The reduced food consumption in females of the 25 mg/kg bw group was not considered adverse.

Summarized results can be found in Attachment 1 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects on brain weight were observed. Altered relative brain weight as observed in male rats at 60 mg/kg bw is considered as a neuro-anatomical endpoint. However, this effect was not observed at higher doses and is therefore considered as incidental.

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes in any treatment group when compared to the control group.

Summarized results can be found in Attachment 1 in the attached background material.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings reported.
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The motor activity was reduced for females in the 150 mg/kg bw group when compared to control animals on Day 1, 8 and 15. It can be noticed that this is associated to a decrease in BW and food consumption. No effects were noted in females of the lower dose groups.
The overall motor activity in male rats was unaffected in all treatment groups.

Summarized results can be found in Attachment 1 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
25 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic effects
Effect level:
60 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
neurotoxicity
Effect level:
150 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects on motor activity were observed up to and including the highest dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
neurotoxicity
Effect level:
60 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects on motor activity were observed at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
neurotoxicity
Effect level:
150 mg/kg bw (total dose)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reduced overall motor activity
Key result
Critical effects observed:
no
Conclusions:
The study was conducted similar to OECD guideline 424 with acceptable restrictions and under GLP conditions. Under the conditions of the test, the NOAEL for systemic toxicity in the rat was 25 mg/kg bw/day. No impairment of motoractivity was found for male rats, while female rats showed signs of reduced overall motoractivity at a dose of 150 mg/kg bw/day. Therefore, the NOAEL for motor activity was 150 mg/kg bw for male rats and 60 mg/kg bw for female rats.
Endpoint:
neurotoxicity: oral
Remarks:
developmental
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Apr 2004 - 18 Jan 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.6300 (Developmental Neurotoxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD® (SD) IGS BR
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd, Margate, Kent, England
- Age at study initiation: 65 days
- Weight at study initiation: Initiation of the study: 180 - 252 g; Day 0 of gestation: 210 - 295 g
- Fasting period before study: no
- Housing: during pregnancy: individually, starting on GD 17 till PND 17: individual dam plus litter in polypropylene stainless steel cages; PND 17 till weaning: individual dam plus litter, from weaning: up to 4 littermates, in stainless steel cages
Acclimatisation (F0): up to 4 female animals in stainless steel TR18 cages with stainless steel grids.
Mating (F0): one female and one male animal in polypropylene RB3 modified cages with stainless steel grids.
Gestation: 1 female in polypropylene RB3 modified cages with stainless steel grids.
Littering (F0): one dam + litter in polypropylene RB3 cages.
Lactation (F0): one dam + litter in stainless steel TR18 cages with stainless steel grids.
From weaning (F1): up to 4 littermates (male and female) in stainless steel TR18 cages with stainless steel grids.
- Diet: standard powdered rodent diet (SDS VRF1 Certified Diet supplied by Special Diet Services, Witham, Essex, England), ad libitum.
- Water: water from the public supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): own supply of filtered fresh air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 May 2004 To: 24 August 2004
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): SDS VRF1 diet, the test substance was incorporated into the ground diet to provide the required concentrations by dilution of an appropriate premix. An initial premix at a concentration of 2300 ppm was prepared.
- Storage temperature of food: stored at -20˚C immediately after preparation. Daily aliquots of diet were removed from the freezer and allowed to thaw out for at least 6.5 hours prior to being offered to the animals, with fresh food fed daily at approximately 16.00 BST.

DIETARY LEVELS
Target test substance concentrations were 20, 45, and 90 ppm
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Information on the homogeneity of mixing, stability and concentration of the test material in the diet were determined as part of this study. The homogeneity and the stability, during ambient temperature storage for 24 hours and frozen storage for 15 days, were assessed in SDS VRF1 diet formulations at nominal concentrations of 20 and 180 ppm.
Single samples of each formulation (nominally 200g accurately weighed) prepared for administration on the first week of treatment and during the first week of lactation were taken for analysis of achieved concentration. Two assays were performed from each sample. Any unused samples were discarded upon issue of the final report.
The mean concentrations of the test item in test diet formulations analysed during the study were within applied limits (+10%/-15% of nominal), with the exception of one low level sample (Week4, 20 ppm) which was 22% below nominal and represented the mean offour analyses.
Duration of treatment / exposure:
F0 females received treated diet from Day 3 after mating to Day 20 of lactation (inclusive).
Frequency of treatment:
continously via the diet
Dose / conc.:
20 ppm
Remarks:
corresponding to an actual ingested dose of 1.6 - 4.1 mg/kg bw/day. See Table 4 under "Any other information on results incl. tables" for details.
Dose / conc.:
45 ppm
Remarks:
corresponding to an actual ingested dose of 3.8 - 9.2 mg/kg bw/day. See Table 4 under "Any other information on results incl. tables" for details.
Dose / conc.:
90 ppm
Remarks:
corresponding to an actual ingested dose of 6.6 - 18 mg/kg bw/day. See Table 4 under "Any other information on results incl. tables" for details.
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
Details on mating procedure (for developmental toxicity study):
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 10 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Observations and clinical examinations performed and frequency:
SERIAL OBSERVATIONS (F0 females)

Clinical observations

Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). In addition, a more detailed physical examination was performed on each F0 animal on Days 0, 7, 13 and 20 after mating and Days 1, 4, 7, 14 and 21 of lactation to monitor general health.

Body weight

The weight of each F0 female was recorded on Days 0, 3, 6, 10, 14, 17 and 20 after mating, then daily until parturition, and on Days 1, 4, 7, 11, 14, 17 and 21 of lactation.

Food consumption

For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled
was recorded for the following periods:
After mating: Days 0-2 and daily from Day 3 to Day 19
During lactation: Daily from Day 1 to Day 20

From these records the mean daily consumption (g/rat/day) was calculated for each animal.

Parturition observations and gestation length

From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were noted.
The duration of gestation was calculated as the time elapsing between the detection of mating and commencement of parturition.


Litter observations:

SERIAL OBSERVATIONS (F1 offspring)

Records made during littering phase

All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter until weaning. The records maintained were as follows:

Litter size

Daily records were maintained of mortality from Day 1 to weaning on Day 21 and on Day 28. On Day 4 of age, litters containing more than ten offspring were culled to ten leaving, whenever possible, five male and five female offspring in each litter.

Sex ratio

The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and on Day 21 of age.

Clinical signs

Daily records were maintained for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate. In addition, a more detailed physical examination was performed on selected offspring on Day 28 of age and then weekly until termination (Days 28, 35, 42, 49, 56 and 63) to monitor general health.

Body weight

Individual offspring body weights were recorded on Days 1, 4 (before culling), 7, 11, 14, 17, 21 and 28 of age and then weekly to termination (Days 28, 35, 42, 49, 56 and 63).

Sexual maturation

F1 males were examined daily from Day 38 of age for balano-preputial separation until separation occurred. Body weight was also recorded on the day of completion of separation. F1 females were examined daily from Day 28 of age for vaginal opening until opening occurred. Body weight was recorded on the day of vaginal opening. Survival indices The following were calculated for each litter: -Post-implantation, survival index, -Live birth index, -Viability index, -Lactation index.
Specific biochemical examinations:
None
Neurobehavioural examinations performed and frequency:
Behavioural assessment (F0 females)

On Days 12 and 18 after mating and on Days 4, 11 and 20 of lactation, twelve females per group were subjected to functional observational battery (FOB, in-the-hand and arena) investigations.

Behavioural assessments (F1 offspring)

Behavioural assessments were performed on the Days indicated for each specific test. The initial allocation of offspring to behavioural tests was made on Day 4 of age based on their within litter identifier. These identifiers were given to the offspring on Day 1 of age. For each litter, animals were randomly identified within each sex, with male offspring numbered first. The number of offspring available for testing was closely monitored and adjustments were made as necessary to the initial selection to ensure as far as possible that at least 10 offspring of each sex were available for each set of tests. In some cases it was necessary to allocate occasional offspring to more than one set of tests.

Functional Observational Battery (FOB) Procedures

On Days 4, 11, 21, 35, 45 and 60 of age, generally up to 12 male and 12 female offspring per group, using one male or one female per litter, were subjected to FOB investigations. On Day 4 of age, 13 male offspring were tested due to insufficient female offspring being available amongst the last litters assessed. The behavioural assessments were customised for the developmental stage being observed. Where possible, the same offspring from each litter was subjected to FOB procedures at the various time points.

Motor activity

Motor activity of the offspring was monitored on Days 13, 17, 22 and 60 of age and measured on each occasion over a one hour recording period. On Day 13, it was recorded whether the eyes were open or closed. Up to 12 male and 12 female offspring per group, using one male or one female per litter, were assessed. Where possible, the same offspring from each litter was used for all the various time points.

Sensory function (quantitative)

Evaluation of sensory functions was assessed by auditory startle habituation and pre-pulse inhibition of startle on Day 23/24 and Day 61/62 of age. One set of up to 12 male and 12 female offspring per group, using one male or one female per litter, were assessed for habituation. A further set of up to 12 male and 12 female offspring per group, using one male or one female per litter, were assessed for pre-pulse inhibition. Where possible, the same offspring from each litter was used for both time points but no offspring was tested for both startle habituation and prepulse inhibition.

Learning and memory

Assessment of learning and memory was performed in the Morris maze from Day 23/24 and Day 61/62 of age and consisted of a series of 3 trials conducted on each of 4 consecutive days. One set of up to 12 male and 12 female offspring per group, using one male or one female per litter, was assessed from Day 23/24. A further set of up to 12 male and 12 female offspring per group, using one male or one female per litter, was assessed from Day 61/62. Occasional offspring may have been subjected to other behavioural assessments during the study but no offspring was tested for learning and memory at both timepoints.

Sacrifice and (histo)pathology:
NECROPSY AND HISTOLOGY

Time of necropsy

For each group excess offspring from each litter, not required following standardisation of the litter size to ten, were killed at Day 4 of age. For each group, F0 females were killed at Day 21 of lactation. On Day 11 of age, either one male or one female offspring from each litter (where possible) was selected for neuropathology (immersion) procedures. As far as possible an equal number of male and female offspring were selected within each group. Following weaning of the litters (Day 21 of age), within each group, either one male or one female was selected, where possible, from each litter for neuropathology necropsy (perfusion) procedures. As far as possible an equal number of male and female offspring were selected within each group.
For each group, up to five of the surviving weaned offspring in each litter were selected for further behavioural assessments. The remaining unselected offspring were retained until Day 28 of age as a contingency against any mortality immediately following weaning and were killed and discarded without examination. For each group, the offspring selected for further behavioural assessments were killed on Day 65 of age. For each litter, either one male or one female was selected for neuropathology necropsy (perfusion) procedures ensuring, as far as possible, an equal number of male and female offspring were selected within each group.
Macroscopic pathology

F0 females

For all animals (Day 21 of lactation), a full macroscopic examination was performed. Any abnormality was recorded. The required tissue samples were preserved in appropriate fixative. This examination included assessment of the number of implantation sites in the uterus. The brain was removed, weighed and fixed in 10 % Neutral Buffered Formalin (NBF).

Day 11 F1 offspring selected for neuropathology necropsy (immersion) procedures

The animals were examined externally and internally for macroscopic abnormalities. Specimens of any abnormal tissues were retained in appropriate fixative. The carcass was then immersed in 10 % Neutral Buffered Formalin for approximately 24 hours, after which the brain was removed and weighed.

Day 21 and Day 65 F1 offspring selected for neuropathology necropsy (perfusion) procedures

Following confirmation of deep anaesthesia or death the heart was exposed to allow perfusion to take place. A needle was inserted into the left ventricle and a small incision made in the right atrium. Following perfusion, the animals were subjected to a macroscopic necropsy examination. The brain was transected from the spinal cord above the first cervical spinal nerve and weight, length and width measurements taken. The length of the brain was measured between the rostral part of the cerebral hemispheres and the most caudual part of the cerebellum, while the width was measured at the widest part of the cerebral hemispheres. The following tissues were removed and retained in fixative (glutaraldehyde and paraformaldehyde):

Day 21 F1 offspring:
Brain
In addition any abnormalities of tissues observed and the carcass were also retained in fixative.

Day 65 F1 offspring:
Brain
Dorsal root fibres – cervical and lumbar
Dorsal root ganglia – cervical and lumbar
Eyes
Optic nerves
Sciatic nerves
Skeletal muscle – gastrocnemius
Spinal cord
Tibial nerves – at knee
Tibial nerves – calf muscle branch (es)
Ventral root fibres – cervical and lumbar

In addition any abnormalities of tissues observed and the carcass were also retained in fixative. For peripheral nerves, only right-sided specimens were removed and left sided specimens were retained in situ.

F1 offspring not selected for neuropathology necropsy (perfusion) procedures

A full macroscopic examination of all animals reaching scheduled termination on Day 65 of age was performed. The exception to this was unselected offspring culled on Day 4 of age, amongst whom only those offspring considered to be externally abnormal were examined. Any abnormality was recorded. The required tissue samples were preserved in appropriate fixative. The brain was removed, weighed and fixed in 10 % Neutral Buffered Formalin (NBF).

Premature death amongst F1 offspring

Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before weaning were examined as detailed above; this also included an assessment for the presence of milk in the stomach, where applicable.

Histopathological processing of tissues

F1 offspring selected for neuropathology necropsy (perfusion) procedures

For all perfusion fixed Day 65 animals, the brain, eyes, optic nerves, skeletal muscle (right gastrocnemius), spinal cord, dorsal root ganglia and dorsal and ventral root fibres and any tissues deemed to be macroscopically abnormal were embedded in paraffin wax. For Day 11 and 21 offspring only the brain and abnormal tissues were retained, therefore only these were processed. For the Control and high dosage group, ten male and ten female offspring at Day 11 and 21 of age (where possible), and ten male and ten female offspring at Day 65 of age per group were selected for further processing. For these animals, sections of the tissues, 4-5 μm in thickness, were processed to slide for selected animals and stained with haematoxylin and eosin.
The sciatic and tibial nerves (at knee, and calf muscle branch(es), right) from Day 65 animals were trimmed at necropsy, transferred to storage buffer and stored at 4 °C prior to embedding in resin. They were sectioned at 2 μm as shown in the following table and stained with toluidine blue.

Tissue sectioning for selected neuropathology F0 females and F1 offspring
Tissue Area sectioned
Brain Coronal sections:
Olfactory lobes
Forebrain
Cerebrum, hippocampus, thalamus, hypothalamus
Cerebrum, tectum, tegmentum
Medulla oblongata
Mid-sagittal sections:
Cerebellum, pons
Spinal cord Transverse and longitudinal sections at cervical and lumbar swellings
Dorsal root ganglia One cervical and one lumbar level
Dorsal root fibres Two longitudinal sections, at one cervical and one lumbar levels
Ventral root fibres Two longitudinal sections, at one cervical and one lumbar levels
Eyes (retina) One longitudinal section of each
Optic nerves One longitudinal section of each
Skeletal muscle
(gastrocnemius) One transverse section
Sciatic nerve (right) Longitudinal and transverse sections at the sciatic notch and the mid-thigh
Tibial nerves (right) Longitudinal and transverse sections at the knee
Longitudinal and transverse sections of calf muscle branch(es)

PATHOLOGY

Tissues from Control and High dose F1 offspring subjected to neuropathology necropsy (perfusion) procedures on Day 21 or Day 65 of age were examined by light microscopy as detailed in the table below. Tissues from Day 11 offspring were processed for contingency purposes. Since no treatment-related findings were observed in Day 21 offspring, these Day 11 tissues were not assessed by light microscopy examination.

Light microscopy examination of tissues
Tissue Areas examined
Brain Coronal sections:
Olfactory lobes
Forebrain
Cerebrum, hippocampus, thalamus, hypothalamus
Cerebrum, tectum, tegmentum
Medulla oblongata
Mid-sagittal sections:
Cerebellum, pons
Spinal cord Transverse and longitudinal sections at cervical (C3-C6) and lumbar (L1-L4) swellings
Dorsal root ganglia One cervical (C3-C6) and one lumbar (L1-L4) level
Dorsal and ventral root One longitudinal section at each of the cervical (C3-C6) and one
fibers lumbar (L1-L4) levels
One longitudinal section at each of the cervical (C3-C6) and one
lumbar (L1-L4) levels
Eyes (retina) One longitudinal section of each
Optic nerves One longitudinal section of each
Skeletal muscle (gastrocnemius) One transverse section
Sciatic nerve (right) Longitudinal and transverse sections at the sciatic notch and the
mid-thigh
Tibial nerve (right) Longitudinal and transverse sections at the knee and calf muscle
branch(es)

For the Day 21 offspring, only the brain was examined.

In addition morphometric measurements were conducted to measure the thickness of the brain at four selected areas:

Neocortex

The distance from the pial surface to the top of the white matter was measured along a line perpendicular to a tangent of the pial surface at the point where the cortex exhibits its greatest thickness.

Hippocampus

The greatest dorsal-ventral thickness of the hippocampus was measured.

Corpus callosum

The thickness of the corpus callosum at the midline was measured.

Cerebellum

The width of the pyramis folia was measured perpendicular to its long axis at the midpoint between its tip and base.

The examining and reviewing pathologist had no prior knowledge of the identity of the treatment groups at the time of the histopathological examination.

In addition to these examinations of scheduled tissues, all tissue abnormalities found for F0 females or Day 21 or 65 F1 offspring selected for neuropathology from all groups killed at schedule termination were processed to slide and subjected to microscopic examination by a pathologist.
Positive control:
None
Statistics:
Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Statistical analyses were performed on the majority of data. All statistical analyses were carried out separately for F0 females and male and female offspring. For all parameters, analyses were carried out using the individual animal or litter as the basic experimental unit as appropriate. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Parental generation:
- 20 and 45 ppm: No effects were observed.
- 90 ppm: pale skin was recorded for 8/24 females during Week 2 and/or 3.

Offspring
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No mortality was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Parental generation
- 20 ppm: No treatment related effects were observed.
- 45 ppm: body weight gain was slightly reduced (25% lower than control) during the first three days of treatment only. Thereafter, body weight gain values during gestation were similar to control.
- 90 ppm: marked reduction (92% lower than control) in body weight gain duringthe first three days of treatment. Recovery of weight gain was observed during Days 6-14 of gestation when weight gains were similar to control. However, the Gestation Day 14 to 17 and Day 17 to 20 body weight gains were 12 - 14% lower than control and attained statistical significance.

Body weight gain values during lactation did not indicate any adverse effect of dietary exposure to the test item at any inclusion level investigated.

Summarized results can be found in Attachment 1 in the attached background material and in Tables 1 and 2 under "Additional information on results incl. tables".

Offspring:
- 20 ppm: No treatment-related effects were observed.
- 45 ppm: body weight values for male offspring on Day 1 of age were marginally but statistically significantly lower (5.7%) than Control, however no significant differences from Control were observed in female offspring. Subsequent weight gain of both sexes to weaning and following the cessation of treatment to Day 63 of age was considered to be essentially similar to Control. Therefore, this effect was not considered adverse.
- 90 ppm: male and female offspring body weight values on Day 1 of age were lower (6-9%) than Control with differences for both sexes attaining statistical significance. Subsequent body weight gain of both sexes was similar to Control up to Day 7 of age, however from Day 7 to Day 11 weight gain in both sexes was slightly lower than in Controls with differences attaining statistical significance for males. From Day 11 to weaning on Day 21 of age lower weight gain was observed in offspring of both sexes; these lower gain values also attained statistical significance. As a result, absolute body weight values for males and females on Day 21 of age were 13% lower than their Control counterparts. Following the cessation of treatment, body weight gain for selected offspring to Day 63 of age was considered to be essentially similar to Control such that differences in absolute body weight values on Day 63 of age were only 4-5% lower than Controls.

Summarized results can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Parental generation
- 20 and 45 ppm: food consumption was lower than control on the first day of treatment. Thereafter, the pattern of food intake did not indicate any adverse effect of dietary exposure during the gestation period.
- 90 ppm: During gestation food consumption values at 90 ppm were lower than control during the first 3 days of treatment with values attaining statistical significance. Thereafter, partial recovery of food intake was apparent with values generally similar or superior to those established prior to treatment for the remainder of gestation. However, intake generally remained slightly lower than control, with the difference greatest during Days 18 to 19 of gestation when intake was 20-25% lower than in controls.

Food intake values during lactation did not indicate any adverse effect.

Summarized results can be found in Attachment 1 in the attached background material and in Table 3 under "Additional information on results incl. tables".
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Parental generation

Functional Observation Battery

There were no definite effects of treatment on performance in the arena at any of the dosages investigated.

At 90 ppm activity counts were noticeably lower than Control on Day 20 of lactation with a concomitant reduction in rearing counts; activity count values attained statistical significance. Other behavioral observations were palpebral closure and/or body tremors. As there was no evidence of a dose response for either observation, with the greatest incidences occurring at 20 ppm and the lowest at 45 ppm, the overall incidence of these signs amongst the adult females on this study was considered to be coincidental and unrelated to treatment.


Offspring

Functional Observation Battery (Arena observation, surface righting effect)

- 20 ppm: No treatment related effects were observed.
- 45 and 90 ppm: On Day 60 of age, mean activity counts amongst female offspring were statistically significantly higher than Controls. However, a relationship between this finding and treatment with the test substance remains unclear, as no statistically significant differences were apparent in low beam (cage floor) or high beam (rearing) activity during the first 6 minutes of automated motor activity monitoring of female offspring on Day 60 of age. In males, the tendency of reduced mean activity counts was also observed at 90 ppm. Other observations in these dose groups were slightly flattened gait but this was not considered treatment related, because the increase of the incidence was only minor.

Motor activity

Activity levels for males and females on Day 22 and Day 60 of age, particularly total activity levels which may be regarded as the best indicator of effects on motor activity, showed no statistically significant inter-group differences and were not considered to indicate any adverse effect of treatment.
A higher number of low beam breaks in males of the 90 ppm group on Day 13 was considered unrelated to treatment as the assessments of motor activity in offspring tend to show high variability and no subsequent similar trend at Day 17 of age was observed.

Auditory startle response habituation

Although little or no habituation was evident amongst males and females on each occasion of testing, there was no obvious effect of treatment with the test substance on the latency to peak amplitude or the peak amplitude of the startle response. The lack of habituation was not considered to be related to the test substance as performance in the Control group was similar to that in treated groups. Habituation in this study was demonstrated during assessment of motor activity on Days 22 and 60 of age and this was unaffected by treatment with the test substance.

Pre-pulse inhibition of startle

On Day 61/62 of age males at 90 ppm showed significantly lower percentage inhibition compared with Control. There was a clear increase in the number of males at this inclusion level showing less than 25 % inhibition (10/12 animals) compared with Controls (4/13 animals).
However, it should be noted that 8/12 animals in the 90 ppm group showed a higher level of inhibition on Day 61/62 compared with that on Day 23/24 compared with 10/10 Controls. Two of the animals in the 90 ppm group which showed a lower level of inhibition on Day 61/62 had shown higher levels of inhibition on Day 23/24 compared with the rest of the animals in the group and Controls.
The performance of males at 45 and 20 ppm and females at all inclusion levels were considered unaffected by treatment.

Learning and memory

During testing from Days 23/24 of age males at the 90 ppm dietary level also showed clear evidence of learning and memory in the Morris water maze, as judged by noticeably shorter trial times, and lower numbers of pool sector entries and failed trials on Day 4 of testing compared with Day 1. However, a slight impairment in the performance of these animals was seen on evaluation Day 4. On Day 4, significantly higher than Control trial times and sector entries were noted for the high dose males. The trial times and sector entry values on this evaluation day exceeded the upper limit of the historical Control range. However, it was concluded that this finding was of no long-term toxicological importance as performance of males at this inclusion level on Day 61/62 of age was similar to Controls. There was no effect of treatment on the performance of male offspring at 45 or 20 ppm or female offspring at any dietary level.
At the Day 61/62 interval overall performance of males at all inclusion levels and females from the 45 ppm and 20 ppm levels was similar to Controls. Females at 90 ppm displayed a slightly inferior performance than female Controls demonstrated by longer mean swimming times on Day 2 of testing and increased number of sector entries on Day 4 of testing attaining statistical significance. The trial times and sector entry values on this evaluation day exceeded the upper limit of the historical Control range (see Table 6 under “Overall remarks, attachments”). There was evidence of learning and memory, as judged by the lower swimming times, and lower number of failed trials and sector entries on Day 3 compared with Day 1, however, no improvement in performance was seen between Days 3 and 4 of testing in contrast to the consistent improvement recorded in the other study groups.

Summarized results can be found in Attachment 1 in the attached background material.
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Parental generation
Measurements of brain weight conducted at necropsy for treated animals were similar to control, did not attain significance and were not considered to indicate any effect of treatment at any of the inclusion levels investigated.

Offspring
not examined

Summarized results can be found in Attachment 1 in the attached background material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Parental generation
- 20 and 45 ppm: No treatment related effects were observed.
- 90 ppm: common findings of enlarged spleen (6/24 animals), pale liver (3/24 animals) and kidneys (2/24 animals), congested mesenteric lymph nodes (4/24 animals) and dark contents within the lower gastro-intestinal tract (5/24 animals) were observed. No similar findings were observed in the control group. Subsequent microscopic examination of these tissues revealed tissue changes of a type and severity commonly seen in rats of this age and strain at this laboratory. However, in view of the clustering of the macroscopic and histopathological findings in females at the high inclusion level, an association with treatment could not be discounted.

Offspring
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in the tissues presented for neuropathological examination which were considered to be related to treatment. All findings were of a type and severity commonly seen in rats of this age and strain at this laboratory. There were no microscopic abnormalities detected in the brain tissue of any animal examined.

Brain morphometry
Intergroup differences in brain morphometry measurements were generally small. On Day 21 of age, at 90 ppm, males presented with slightly larger hippocampus and females with slightly larger neocortex, with differences attaining statistical significance. Such differences were not apparent at Day 65 of age therefore these findings were considered to be of no long-term toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of the tissues showing abnormalities in the gross pathological examination revealed tissue changes of a type and severity commonly seen in rats of this age and strain at this laboratory. However, in view of the clustering of the macroscopic and histopathological findings in females at the high inclusion level, an association with treatment could not be discounted.

Summarized results can be found in Attachment 1 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
no effects observed
Description (incidence and severity):
Litter data
Total and live litter size was unaffected by treatment.

Offspring survival
No treatment related effects were observed.

Sex ratio
Sex ratio was similar in all groups.

Sexual maturation
No treatment-related effects were observed.

Summarized results can be found in Attachment 1 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal systemic toxicity
Effect level:
45 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual ingested dose of 3.8 - 9.2 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
maternal systemic toxicity
Effect level:
90 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Remarks on result:
other: corresponding to an actual ingested dose of 6.6 - 18 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
morphological development
Effect level:
90 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level.
Remarks on result:
other: corresponding to an actual ingested dose of 6.6 – 18 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
functional development
Effect level:
20 ppm
Based on:
test mat.
Remarks:
maternal dose during gestation and lactation
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual ingested dose of 1.6 - 3.8 mg/kg bw/day.
Key result
Dose descriptor:
LOAEL
Remarks:
functional development
Effect level:
45 ppm
Based on:
test mat.
Remarks:
maternal dose during gestation and lactation
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: effects in FOB (Arena observation-surface righting effect in males and females) starting at 45 ppm; reduced pre-pulse inhibition of the startle response (males), slightly impaired performance in the Morris water maze (males) at 90 ppm
Remarks on result:
other: corresponding to an actual ingested dose of 3.8 - 9.2 mg/kg bw/day.
Key result
Critical effects observed:
no

Table 1: Body weight - group mean values (g) for females during gestation (F0)

Dose

(ppm)

Day

0

3

6

10

14

17

20

0

Mean

254

275

288

309

332

365

408

SD

13

13

13

14

15

21

31

n

24

24

24

24

24

24

24

20

Mean

254

273

285

306

328

363

411

SD

17

18

18

22

23

24

29

n

24

24

24

24

24

24

24

45

Mean

252

273

281

303

325

357

400

SD

15

14

14

18

18

19

21

n

24

24

24

24

24

24

24

90

Mean

251

272

273**

293**

316**

345**

382**

SD

17

16

17

18

20

22

27

n

24

24

24

24

24

24

24

Significant difference to Control: **p<0.01

 

Table 2: Body weight change - group mean values (g) for females during gestation (F0)

Dose

(ppm)

Days

0-3

3-6

3-10

3-14

3-17

3-20

6-10

10-14

14-17

17-20

0

Mean

22

12

34

56

89

132

21

23

33

43

SD

5

4

6

8

15

25

7

6

11

12

n

24

24

24

24

24

24

24

24

24

24

20

Mean

19

12

33

55

90

137

21

22

35

48

SD

5

5

9

10

10

16

8

6

5

8

n

24

24

24

24

24

24

24

24

24

24

45

Mean

21

9*

30

52

84

128

21

22

32

43

SD

5

5

7

10

11

13

7

6

5

6

n

24

24

24

24

24

24

24

24

24

24

90

Mean

21

1**

21**

44**

73**

110**

20

23

29*

37**

SD

7

6

9

11

12

17

8

6

5

8

n

24

24

24

24

24

24

24

24

24

24

Significant difference to Control: *p<0.05, **p<0.01

 

Table 3: Food consumption - group mean values (g/rat/day) for females during gestation (F0)

Dose

(ppm)

Days

0-2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

0

Mean

27

29

29

29

29

31

30

30

31

32

32

31

31

33

33

30

36

33

SD

3

4

4

2

3

4

4

2

3

3

3

3

3

3

3

5

8

8

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

20

Mean

27

26**

27

28

28

29

30

30

30

31

31

29

31

31

34

29

32

32

SD

2

2

3

3

3

3

3

3

3

3

3

3

3

3

4

5

4

6

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

45

Mean

28

24**

29

30

30

30

30

31

30

32

32

31

31

32

34

32

33

31

SD

3

3

3

3

3

3

3

3

4

3

4

3

4

3

6

8

6

6

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

90

Mean

27

20**

24**

26**

28

28*

28*

28*

29

30**

30*

30

30

31**

30*

26*

29**

25**

SD

3

3

3

5

4

3

3

3

3

3

4

3

3

3

4

3

4

4

n

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

Significant difference to Control: *p<0.05, **p<0.01

 

Table 4: Achieved dosage - group mean values (mg/kg/day) during gestation and lactation (F0)

Dose

(ppm)

Days of gestation

Days of lactation

3-5

6-9

10-13

14-16

17-19

1-3

4-6

7-10

11-13

20

1.9

2.0

1.9

1.9

1.6

2.8

3.3

3.8

4.1

45

4.5

4.7

4.5

4.3

3.8

7.2

7.5

8.5

9.2

90

7.8

9.0

8.7

8.2

6.6

12.9

15.0

16.7

18.0
Conclusions:
The study was conducted according to guideline EPA OPPTS 870.6300 and under GLP conditions.

The No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 45 ppm (3.8 to 9.2 mg/kg/day) based on effects on body weight, body weight gain, food consumption and clinical signs of toxicity seen in treated dams receiving a dietary concentration of 90 ppm (6.6 to 18 mg/kg/day). A dietary concentration of 90 ppm (achieved dosage range 6.6 to 18.0 mg/kg/day) was considered to be the clear NOAEL for the morphological development of the nervous system in the CD rat. And a dietary concentration of 20 ppm (achieved dosage range 1.6 to 3.8 mg/kg/day) was considered the NOAEL for functional development of the nervous system in the CD rat, based on FOB disturbances at higher doses.
Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Sep - 10 Dec 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Version / remarks:
adopted in 1997
Deviations:
yes
Remarks:
methodological limitations (rats instead of hens; Tests were performed on weeks -1, 4, 8, 13 and not once a week;histopathology was performed on solely 6 rats from control dose and maximal dose; no biochemical measurements (NTE assay) were performed)
Qualifier:
according to guideline
Guideline:
other: US-EPA FIFRA, Guideline 82-5
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: : Harlan Sprague Dawley, Inc. Indianapolis, IN, USA
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 252 - 287.6 g (males), 167 - 194.6 g (females)
- Fasting period before study: not applicable
- Housing: individually in stainless steel, wire mesh cages
- Diet: ground, certified Rodent Chow #5002 (Purina Mills, Inc.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 Sep 1992 To: 10 Dec 1992
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (offered daily)
- Mixing appropriate amounts with (Type of food): Test diets were prepared by direct addition of the test substance to ground rodent feed. A premix was prepared to ensure homogeneous distribution of the test material in the diet.
- Storage temperature of food: frozen
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance in the diet was analyzed throughout the study. Stability of the test substance in the diet (30 and 500 ppm) was determined to be at least 22 days in closed polyethylene containers stored frozen and at room temperature to be at least 14 days in open glass feed jars. Concentration verifications of the test diets showed analytical values ranging from 90.8 - 107.2% of the nominal dietary concentrations with means of 29.78, 119 and 488 ppm for the 30, 125 and 500 ppm groups, respectively. Homogeneity was tested by sampling from the top, middle and bottom of the mixing bowl for all dose groups.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continously via the diet
Dose / conc.:
30 ppm
Remarks:
corresponding to an actual ingested dose of 1.5-2.3 and 1.8-2.6 mg/kg bw/day for males and females, respectively.
Dose / conc.:
125 ppm
Remarks:
corresponding to an actual ingested dose of 5.9-10.2 and 6.9-10.1 mg/kg bw/day for males and females, respectively.
Dose / conc.:
500 ppm
Remarks:
corresponding to an actual ingested dose of 22.8-40.2 and 21.6-43.9 mg/kg bw/day for males and females, respectively.
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results reported for a 104 week chronic study (Report Number HLA 6111-113) and a 13 week subchronic study (Report Number HLA 6111-110). The high dietary concentration of 500 ppm was selected because it was anticipated to induce significant body weight effects. The mid dose of 125 ppm was anticipated to produce some but less severe toxicity and 30 ppm was anticipated to be a clear no effect level.
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly, and on the days of neurobehavioural evaluations, and prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked: cage posture, convulsions, handling reactivity, tremors, vocalization, palpebral closure, unusual behaviour, gait, body position, breathing pattern, arousal, defecation, urination, rears, approach response, startle response, tail pinch response, pupil size, muscle tone, piloerection, lacrimation, salivation, exophthalmus, emaciation, dehydration, fur appearance, crusts, visual placing, grip strength, body temperature, body weight, air righting, hind leg splay.
- Description of procedures: not reported
- Minimization of bias: yes
- Same technicians used throughout testing: No (the same technicians observed the male animals at all observation time points, the female animals were observed by mostly 2 technicians)
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: pre-study (Week -1), Week 4, 8, 13
- Number of animals examined: 10 animals/sex and dose
- Environmental conditions: polycarbonate shoebox style cages
- Noise level: white noise, 59 - 62 dBA
- Scoring criteria: the scoring is described in Attachment 1 in the attached background material.
- Duration of observation period for open field observations: not reported

LOCOMOTOR ACTIVITY: Yes
- Replicates used: not reported
- Type of equipment used: not reported
- Length of session, number and length of subsessions: 90 min with intrasession intervals of 10 min
- Parameters measured: ambulatory activity, fine motor activity, rearing, and the sum of these individual types of activity
- Number of animals examined: all animals of all groups

Sacrifice and (histo)pathology:
- Time point of sacrifice: during the 14th week of treatment
- Number of animals sacrificed: 10/sex/dose (at the final day of sacrifice, all surviving animals were sacrificed).
- Parameters measured: All animals scheduled for neuroanatomic pathology were given an abbreviated necropsy examination consisting of examination of the thoracic and peritoneal cavities.
- Brain weight: yes
- Length and width of brain: no
- Procedures for perfusion: Animals were anesthetized with sodium pentobarbital (i.p.) and perfused in situ by intracardiac perfusion. After in situ perfusion the calvaria and the vertebral arches covering the brain and spinal cord were removed, and the peripheral nerves in the hind limb were exposed. The tissues were fixed in neutral buffered formalin.
- Number of animals perfused: 10 animals/sex/dose
- Tissues evaluated: brain, spinal cord, gasserian ganglion, nerve roots, and dorsal root ganglia, peripheral nerves (sciatic, peroneal, sural, and tibal), tail (for identification).
- Type of staining: hematoxylin and eosin as well as luxol fast blue and Bielschowsky's techniques
- Methodology of preparation of sections: not reported
- Thickness: 5 - 6 µm
- Embedding media: paraffin
- Number of sections: not reported
- Number of animals evaluated from each sex and treatment group: 6 animals/sex from control and high-dosage group

Sections of the peripheral nerves (sciatic, peroneal, sural and tibial) were embedded in glycol methacrylate, sectioned at 2 µm and stained with hematoxylin and eosin, toluidine blue and Bielschowsky's techniques.
Other examinations:
None
Positive control:
None
Statistics:
Quantitative continuous variables: Levene's test for equality of variance, analysis of variances (ANOVA) and t-tests were performed.

Motor activity: nested analysis using repeated measures analysis of variance with dose as the grouping factor and test period and intrasession interval as within subject factors. The epsilon-adjustment procedure (Greenhouse-Geisser correction) was used in the repeated measures analysis. Total cumulative test session activity for each testing interval was evaluated as described above for parametric data.

Incidence data: Fisher's Exact test was used in general, further tests were Gamma, Kendall's Tau-B, Stuart's Tau-C and Somer's D measures of association.

For all statistical tests except neuropathology frequency comparisons, the probability value of < 0.05 (two-tailed) was used as the critical level of significance. A probability value of < 0.05 (one-tailed) was used as the critical level of significance for neuropathology frequency comparisons.

Unless otherwise stated, comparisons were made to the control group. Concentration verification analyses of the tests diets showed analytical values ranging from 90.8 to 107.2 % of nominal values.
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed during the study in any animal.

Summarized results can be found in Attachment 2 in the attached background material.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No animal died before the final sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 30 ppm: No treatment-related effects were observed.
- 125 ppm: reduced body weight gain in males during Week 1. In Week 2 a compensatory increase was observed. Decreased body weights and/or body weight gains were observed occasionally after Week 2 in females.
- 500 ppm: Body weight was decreased (6-7%) in both sexes during Week 1. In Week 2 a compensatory increase was observed, but body weights continued to be statistically significantly lower throughout the study.

Summarized results can be found in Attachment 2 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was dose-dependently decreased. During Week 1 food consumption of males was reduced by 4, 16, and 47% in the 30, 125, and 500 ppm dose group, respectively. For females food consumption was reduced by 16 and 53% in the 125, and 500 ppm group, respectively. This general pattern persisted throughout the study, although the differences observed at 125 and 30 ppm in males were less consistent and of smaller magnitude (about 4 - 8%) than those observed in the 500 ppm males (9 - 16%). In females, decreases of food consumption of 9 - 12% were occasionally seen for the 125 ppm group. In the high dose females decreases of 9 - 17% were consistently observed during the study.
The reduction in food consumption was, in part, attributed to palatability.

Summarized results can be found in Attachment 2 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 30 ppm: No effects were observed.
- 125 ppm: In females of the relative brain weights were statistically significantly increased.
- 500 ppm: Brain weights of high dose males and females were statistically less than controls. The relative brain weights were significantly increased.

These findings were considered an effect of the test substance on the overall growth, rather than a specific effect on the nervous system.

Summarized results can be found in Attachment 2 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related effects.

Summarized results can be found in Attachment 2 in the attached background material.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No treatment related effects.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related gross findings at any dose or microscopic findings of the central or peripheral nervous system tissues in the high dose treatment group.

Summarized results can be found in Attachment 2 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The FOB did not reveal a consistent or striking profile of effects, which could be considered as indicative for neurotoxicity. However, males in the high-dose group showed an increase in the number of rears, and an increased incidence of hyperactivity during Week 8 and 13. Because males tend to have a low level of activity under these test conditions relative to females, it is difficult to discern the effects of test substances, which depress activity. Similar but less conclusive signs were observed in the females. Statistically significant increases in the number of rears and the incidence of hyperactivity were also detected in females at 30, 125, and 500 ppm during Week 8.
Due to the slight magnitude of the changes, their transient nature, and the lack of supporting evidence from the automated motor activity assessments, the findings are equivocal.


Motor-activity: There were no significant treatment-related differences observed in both sexes.

An overview of significant functional observational battery findings can be found in the table under "Any additional information on results incl. tables".

Summarized results can be found in Attachment 2 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to an actual ingested dose of 1.5-2.3 and 1.8 - 2.6 mg/kg bw/day for males and females, respectively.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
125 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to an actual ingested dose of 5.6 - 10.2 and 6.9 - 10.1 mg/kg bw/day for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
neurotoxicity
Effect level:
125 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose-level.
Remarks on result:
other: corresponding to an actual ingested dose of 5.6 - 10.2 and 6.9 - 10.1 mg/kg bw/day for males and females, respectively.
Key result
Dose descriptor:
LOAEL
Remarks:
neurotoxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
Remarks on result:
other: corresponding to an actual ingested dose of 22.8 - 40.2 and 21.6 - 43.9 mg/kg bw/day for males and females, respectively.
Key result
Critical effects observed:
no

Overview of significant functional observational battery findings:

 Time exposure        Week 4        Week 8        Week 13
 Group (ppm)  30  125  500  30  125  500  30  125  500
 Males                          
 rears           i      i
 arousal           i      ia 
 body weight     d      d     d 
 Females                          
 rears       ib  ib  ib       
 arousal       ib ib  ib       
 forelimb grip strength    db db             
 body weight    d d       d    d  d

statistically significant increased (i) or decreased (d) compared to control group

a: not statistically significant, however, considered to be biologically significant

b: the biological significance is questionable

 

 

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
The study was conducted similar to OECD guideline 424 and under GLP conditions. Rats received dietary concentrations of 30, 125 and 500 ppm of the test substance over a period of approximately 13 weeks. There were no clinical signs of toxicity, equivocal FOB findings, and no motor activity changes observed in this study, which were attributed to treatment with the test substance. There were also no gross pathological or microscopic findings for high dose animals. At 500 ppm administration of the test substance over a period of 90 days caused adverse effects on body weight, body weight gain, and food consumption. Under the conditions of the test, the NO(A)EL for neurotoxicity was 125 ppm (corresponding to an actual ingested dose of 5.6 - 10.2 and 6.9 - 10.1 mg/kg bw/day for males and females, respectively). The NO(A)EL for systemic toxicity was 30 ppm (1.5-2.3 and 1.8 - 2.6 mg/kg bw/day for males and females, respectively).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
With respect to neurotoxicity, an acute, a subchronic and a developmental neurotoxicity study are available. All three studies are guideline studies, conducted under GLP conditions. Thus, they are considered of reliable quality and validity, fulfilling the criteria of key studies.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Please refer to the respective endpoint summary. 

Additional information

With respect to neurotoxicity, an acute, a subchronic and a developmental neurotoxicity study are available. All three studies are guideline studies, conducted under GLP conditions. Thus, they are considered of reliable quality and validity, fulfilling the criteria of key studies. In addition, a developmental neurotoxicity study is available.

 

Neurotoxicity: oral administration

Data on neurotoxicity are available after single and repeated exposure to characterize the potency of the test substance to induce neurotoxicity after short-term and subchronic exposure. Moreover, a neurotoxicity developmental toxicity study was conducted with the test substance. All studies were conducted with rats.

 

In all key studies, rats showed loss of body weight and/or body weight gain at higher doses, often with decreased food consumption. No severe clinical signs of toxicity were observed in any study.

In the acute neurotoxicity key study (Report number CTL/AR7090), the motor activity was reduced in females in the 150 mg/kg bw group (highes dose tested) when compared to control animals on Day 1, 8 and 15 after treatment. No effects were noted in females of the lower dose groups or males of any dose group. The systemic NOAEL was set at 25 mg/kg bw based on reduced body weight (gain) and food consumptions in males and females of higher dose groups. The NOAEL for neurotoxicity was 60 mg/kg bw for females and 150 mg/kg bw for male rats.

 

In a supporting acute neurotoxicity study (Report number 91N0126), decreased body weights were observed in males and females of the high dose group (600 mg/kg bw), some of these changes were transient. Signs of neurotoxicity were apparent at 150 and 600 mg/kg bw/day but were mostly transient.

 

In the subchronic toxicity study (Report number 91N0127), decreased body weights were noted. No neurotoxic effects were observed. Minimal FOB findings and absolute brain weight changes were recorded. A possible excitatory effect on activity was observed in the open-field at 500 ppm (the highest dose tested). No further effects on the nervous system were observed.

Based on the observed effects, a NOAEL for systemic toxicity was set at 1.5 mg/kg bw and for neurotoxicity of 5.9 mg/kg bw. The systemic NOAEL in this study was 30 ppm, corresponding to 1.5 – 2.3 mg/kg bw/day for males and 1.8 – 2.6 mg/kg bw/day for females, the NOAEL for neurotoxicity was 125 ppm, corresponding to 5.6 – 10.2 mg/kg bw/day for males and 6.9 – 10.1 mg/kg bw/day for females.

 

Additionally, a developmental neurotoxicity study was conducted (Report number PFX 004/042634) in Sprague Dawley rats, in which dams were administered the test substance via the diet during gestation and lacrimation. No adverse treatment-related maternal effects were observed at 20 ppm. At 45 ppm body weight gain was slightly reduced during the first 3 days of treatment while food consumption was reduced on the first day of treatment at 45 and 20 ppm. There was no adverse effect of treatment at any inclusion level on the numbers of implantations, litter size or offspring survival to weaning. Effects on body weight and body weight gain in the offspring were largely confined to the 90 ppm group, the same inclusion level where marked effects on body weight gain had been recorded for the parent females. At this inclusion level, mean body weights of male and female offspring on Day 1 of age were reduced compared to control, and reduced weight gains were recorded from Day 7 to 21 of age. After weaning, body weight gain was essentially similar to Controls such that by Day 63 body weights were only 4-5% reduced compared to controls. At 45 ppm mean body weights of male offspring were marginally reduced compared to Controls on Day 1 of age, however weight gain of both sexes through to Day 63 of age were essentially similar to control. Offspring body weights were unaffected by treatment at 20 ppm.

The mean age of completion of vaginal opening in female offspring and balano-preputial separation in male offspring were unaffected by treatment.

Treatment related effects on the arena observations of offspring were confined to differences in the surface righting reflex of males in the 90 ppm group on Day 11 of age and levels of activity among females in the 90 and 45 ppm groups on Day 60 of age. In isolation, the effect on surface righting is considered not to be an adverse effect on the functional development of the offspring, and the apparent higher levels of activity amongst females on Day 60 of age were not confirmed by automated motor activity monitoring of the same animals on the same day. However, following a conservative approach, the effect on surface righting was considered for setting the effect level for neurotoxicty.

Motor activity of the male and female offspring were considered to be unaffected by treatment. On Day 13 of age, males at 90 ppm showed higher activity with low beam breaks (cage floor activity) attaining statistical significance. However the activity of these animals was comparable to Control on Day 17 of age, where decreased high beam breaks (rearing activity) was evident. In the absence of any consistency between these observations these differences were considered to be coincidental and unrelated to treatment. More importantly these differences did not persist and were not apparent at testing on Days 22 and 60 of age, confirming no long term effect on offspring activity.

The auditory startle response was assessed on Day 23/24 and Day 61/62 of age and there was no obvious effect of treatment on the latency to peak amplitude or the peak amplitude of the startle response. Little or no habituation was evident amongst males and females on each occasion of testing, however habituation in this study has been demonstrated during assessment of motor activity. Moreover, habituation is regarded as the simplest form of learning and more complex cognitive learning was clearly demonstrated during assessment of the offspring in the Morris water maze. The level of habituation recorded among adult male and female rats on Day 61/62 was lower than previously recorded in this laboratory; the reason for this finding is unknown. The level of habituation recorded among male and female rats on Day 23/24 of age was lower than on the validation study HLS/275, but habituation has proved difficult to consistently demonstrate in young pups. The lack of habituation during the assessment of the auditory startle response was therefore considered not to impair the ability of the study to detect developmental effects on learning and memory.

At 90 ppm there was a slight impairment of performance of males from Day 23/24 of age in the Morris water maze, but this difference was concluded to be of no long-term toxicological importance since performance of males from Day 61/62 of age was similar to Controls. In females, a statistically significant difference in performance was observed at 90 ppm at the age of 61/62 days. Although the differences observed in the Morris water maze test do not clearly indicate an adverse effect, the observations in the Morris maze test were considered for setting the effect level for neurotoxicty, following a conservative approach.

 

At 90 ppm the percentage of pre-pulse inhibition of the startle response among males was reduced compared to Controls on Day 61/62 of age but a relationship to treatment remains open, as in Controls, the majority of animals showed a greater level of inhibition compared with that on Day 23/24. There was also a suggestion of slightly impaired performance of females from Day 61/62 of age in the Morris water maze between Days 3 and 4 of testing. No similar effects were observed at testing of the offspring shortly after weaning and there were no histopathological alterations observed in the nervous system at this inclusion level. However, performance on Day 4 of testing was outside the background Control range and an effect of treatment could not be discounted. Therefore this was considered to preclude this inclusion level as a No-Observed-Adverse-Effect-Level (NOAEL) for functional development of the nervous system. No similar findings were apparent at 45 or 20 ppm.

There was not considered to be any clear adverse effect of treatment on the morphological development of the nervous system in the offspring at any inclusion level investigated. Based on the results of this study it was concluded that treatment at 90 ppm was not associated with any selective developmental neurotoxicity. There appeared to be differences in the pre-pulse inhibition of the auditory startle response of males at Day 61/62 of age and a slight impairment of the performance of females in the Morris water maze at the same age, suggestive of an influence of treatment. These findings occurred at an inclusion level associated with reduced offspring body weights on Day 1 of age compared with Controls along with reduced weight gains from Day 7 to Day 21 of age. In addition, parent females at this inclusion level displayed a marked initial reduction in body weight gain with a concomitant reduction in food consumption, reduced body weight gains for the gestation Days 14-17 and Days 17-20 intervals and clinical signs during lactation. The No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 45 ppm (3.8 to 9.2 mg/kg/day based on effects on body weight, body weight gain, food consumption and clinical signs of toxicity seen in treated dams receiving a dietary concentration of 90 ppm (6.6 to 18 mg/kg/day) A dietary concentration of 90 ppm (achieved dosage range 6.6 to 18.0 mg/kg/day) was considered to be the clear NOAEL for the morphological development of the nervous system in the CD rat. A dietary concentration of 20 ppm (achieved dosage range 1.6 to 3.8 mg/kg/day) was considered to be the NOAEL for functional development of the nervous system following a rather conservative approach based on slight effects in FOB (arena observation-surface righting effect) starting at 45 ppm and reduced pre-pulse inhibition of the startle response in males and slightly impaired performance in femlas at 90 ppm in the Morris water maze.

Based on the results of this study it was concluded that treatment at 90 ppm was not associated with any selective developmental neurotoxicity. There appeared to be differences in the pre-pulse inhibition of the auditory startle response of males at Day 61/62 of age and slight impairment of the performance of females in the Morris water maze at the same age, suggestive of an influence of treatment. These findings occurred at an inclusion level associated with reduced offspring body weights on Day 1 of age compared with Controls along with reduced weight gains from Day7 to Day 21 of age. In addition, parent females at this inclusion level displayed a marked initial reduction in body weight gain with a concomitant reduction in food consumption, reduced body weight gains for the gestation Days 14-17 and Days 17-20 intervals and clinical signs during lactation.

 

Overall, the conducted studies revealed clinical signs indicative for neurological effects with brain weight changes in the subchronic and developmental studies. Most effects occured in the presence of systemic and/or maternal toxicity. Thus, although a neurotoxic potential cannot be excluded, the observed effects are not severe enough to trigger specific classification, which is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008, index number 006 -005 -00 -4).

Justification for classification or non-classification

The available data on neurotoxicity of the test substance do not meet the criteria for classification and labelling according to the CLP Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification. This is in line with the harmonised classification of the test substance (Annex VI of Regulation (EC) 1272/2008, index number 006 -005 -00 -4).