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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 2009 - Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study performed for the ultimate degradation product
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Jan 2009 - Nov 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study performed for the ultimate degradation product
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: appr. 12 wks
- Weight at study initiation: (P) Males: 372-450 g; Females: 205-272 g
- Fasting period before study: no
- Housing: in groups of five
- Diet (e.g. ad libitum): ad-libitum
- Water (e.g. ad libitum): ad-libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55+/- 15 %
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 42 (recovery + 14 days)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 15, 30, 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): --
- Purity: distilled
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of N-Butylpyridinium Chloride in the test material formulations was determined by high performance liquid chromatography (HPLC).
The test material formulations were diluted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Procedure:
HPLC: Agilent Technologies 1050/1200, incorporating autosampler and workstation
Column: Sonoma 5µ C 18 (250 x 4.6 mm id)
Mobile phase: 0.1% orthophosphoric acid: methanol (90:10 v/v)
Flow-rate: 1 ml/min
UV detector wavelength 259 nm
Injection volume: 25 µL
Retention time: - 3.3 mins
Duration of treatment / exposure:
42 days
Frequency of treatment:
daily
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Dose level Male Female
-------------------------------------------------
0 10 + 5 (recovery) 10 + 5 (recovery)
75 10 10
150 10 10
300 10 + 5 (recovery) 10 + 5 (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose level Male Female
-------------------------------------------------
0 10 + 5 (recovery) 10 + 5 (recovery)
75 10 10
150 10 10
300 10 + 5 (recovery) 10 + 5 (recovery)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before dosing, soon after dosing, and one hour after dosing and then daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing, soon after dosing, and one hour after dosing and then daily

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 and then weekly (males) and Days 0, 7, 14 and 20 post coilum(females)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
During the maturation period, weekly food consumption was recorded for each cage of
non-recovery adults. This was continued for males after the mating phase. For females
showing evidence of mating, food consumption was recorded for the periods covering
Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded
on Days 1 and 4 post partum. Food consumptions were performed weekly for each cage
of recovery adults throughout the study period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Water intake was observed daily by visual inspection of water bottles for any overt
changes.
- Time schedule for examinations:

OTHER:

- Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals
were observed for signs of functionallbehavioural toxicity. Functional performance tests
were also perfonmed on five selected males and females from each non-recovery dose
level, prior to termination , together with an assessment of sensory reactivity to various
stimuli.

- Behavioural Assessment:
Detailed individual clinical observations were performed for each animal using a
purpose-built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymatation
Hyper/Hypothermia
Skin co lour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation
This test was developed from the methods used by Irwin (1968) and Moser et./ (1988).

-Functional Performance Tests
-Sensory Reactivity
-Laboratory Investigations (Hematology, Blood Chemistry, Urinalysis)
-Reproduction Screening (Mating, Pregnancy and Parturition, Litter data, Physical Development)
-Pathology (Organ weights, Histopathology)





Sacrifice and pathology:
yes
Statistics:
Standard statistical methods have been applied for data processing.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One non-recovery female treated with 300 mg/kg/day was killed in extremis on Day 37. Two further non-recovery females from this treatment group were found dead on Days 14 and 39 and one 300 mg/kg/day recovery male was also found dead and cannibalised on Day 56. One control female was killed in extremis on Day 37. There were no further unscheduled deaths during the study.

Animals of either sex treated with 300 and 150 mg/kg/day and females treated with 75 mg/kg/day showed episodes of increased salivation immediately post or one hour post dosing. Hunched posture and lethargy were evident in one 300 mg/kg/day male on Day 21 and one female from this treatment group also showed increased lachrymation on Day 38. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated from all treatment groups throughout the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically significant effects were detected in bodyweight
development.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating. There were no treatment-related effects on mating or conception rates for
animals of either sex treated with 300, 150 or 75 mg/kg/day.
Fertility. There were no treatment-related effects on conception rates.
Gestation Length. There were no differences in gestation lengths. The distribution for
treated females was comparable to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males treated with 300 mg/kg/day showed a statistically significant
increase in liver weight both absolute and relative to terminal bodyweight. Females from
this treatment group also showed a statistically significant increase in relative liver
weight. No such effects were detected in animals of either sex treated with 150 or
75 mg/kg/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related macroscopic abnormalities were detected for surviving
animals of either sex treated with 300 mg/kg/day, animals of either sex treated with
150 or 75 mg/kg/day or surviving recovery animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
SALIVARY GLAND: A higher incidence of lower grades of secretory activity of the
submaxillary salivary glands was seen in relation to treatment for animals of either sex
treated with 300 mg/kg/day or at 150 mg/kg/day and also for females treated with
75 mg/kg/day. Treatment-related changes in secretory activity of the salivary glands can
be regarded as adaptive.
The condition was observed to have regressed among Recovery 300 mg/kg/day males
fo llowing an additional fourteen days without treatment, but a marginal residual effect on
secretion remained for Recovery 300 mg/kg/day females.

BONE MARROW: A higher incidence of higher grades of severity of adipose infiltration
of the bone marrow, indicative of reduced cellularity, was seen for males only treated
with 300 mg/kg/day. A similar effect was not seen for females or for males from any other
treatment level.
The condition had regressed among Recovery 300 mg/kg/day males after completion of
the recovery period.

OTHER FINDINGS (PARENTAL ANIMALS)
Key result
Dose descriptor:
NOEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Critical effects observed:
no
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
salivary glands
Conclusions:
The oral administration of the test material to rats by gavage, at dose levels of 75, 150 and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated at 300 and 150 mg/kg/day and in females treated with 75 mg/kg/day. A 'No Observed Effect Level' (NOEL) for systemic toxicity for females has therefore not been established however the 'No Observed Effect Level' (NOEL) for systemic toxicity for males was considered to be 75 mg/kg/day. The effects detected at 150 mg/kg/day and in 75 mg/kg/day females were confined to adaptive changes in the salivary glands. In the absence of any associated changes these effects were considered not to represent an adverse health effect. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 150 mg/kg/day. No treatment-related effects were detected in the reproductive parameters measured. The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 300 mg/kg/day.
Executive summary:

Study Design

The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996).

Methods

The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SO) IGS BR strain rats. for up to fortyfive consecutive days (including a two week maturation phase. pairing. gestation and early lactation for females). at dose levels of 75. 150 and 300 mglkg/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Two recovery groups, each of five males and five females. were treated with the high dose (300 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioural assessments, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition. haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Pairing of animals within each non-recovery dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each nonrecovery dose group after the completion of the mating phase, and for five selected parental females from each non-recovery dose group on Day 4 post partum.

Non-recovery males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post parium. All non-recovery animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Recovery animals were treated according to the dose group continuously up to the point of sacrifice of the non-recovery males at which time the treatment was discontinued. After fourteen days without treatment, the recovery males and females were subjected to a gross necropsy examination of selected tissues.

Results

Adult Responses:

Mortality

One non-recovery female treated with 300 mg/kg/day was killed in extremis on Day 37. Two further non-recovery females from this treatment group were found dead on Days 14 and 39 and one 300 mg/kg/day recovery male was also found dead and cannibalised on Day 56. One control female was killed in extremis on Day 37. There were no further unscheduled deaths during the study.

Clinical Signs

Animals of either sex treated with 300 and 150 mg/kg/day and females treated with 75 mg/kg/day showed episodes of increased salivation immediately post or one hour post dosing. Hunched posture and lethargy were evident in one 300 mg/kg/day male on Day 21 and one female from this treatment group also showed increased lachrymation on Day 38. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated from all treatment groups throughout the study.

Behavioural Assessment

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Bodyweights

No toxicologically significant effects were detected in bodyweight development.

Food Consumption and Food Efficiency

No adverse effects in food consumption or food efficiency were detected.

Water Consumptions

Males treated with 300 and 150 mg/kg/day showed a substantial increase in water consumption from initiation during Week 3 until the end of the treatment period. Recovery 300 mg/kg/day animals continued to show an increase in water consumption during the first week of the recovery period. Females treated with 300 mg/kg/day showed an increase in water consumption during gestation. No such effects were detected in females treated with 150 mg/kg/day or animals of either sex treated with 75 mg/kg/day.

Haematology

Males treated with 300 mg/kg/day showed a statistically significant increase in haemoglobin and haematocrit at the Day 42 evaluations. No such effects were detected in females treated with 300 mg/kgl/day or animals of either sex treated with 150 or 75 mg/kg/day.

Blood Chemistry

Males treated with 300 mg/kg/day showed a statistically significant increase in aspartate aminotransferase at both the Day 14 and Day 42 evaluations. Females from this treatment group also showed an increase in aspartate aminotransferase at the Day 4 post parium evaluations. Following fourteen days without treatment, recovery 300 mg/kg/day males continued to show an increase in aspartate aminotransferase. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg/day.

Urinalysis

There were no treatment-related effects on urinalytical values measured.

Reproductive Performance

Mating.There were no treatment-related effects on mating or conception rates for animals of either sex treated with 300, 150 or 75 mg/kg/day.

Fertility.There were no treatment-related effects on conception rates.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses

Offspring Litter Size and Viability.Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls.

Offspring Growth and Development.
Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls.

Litter observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology

Necropsy.No treatment-related macroscopic abnormalities were detected for surviving animals of either sex treated with 300 mg/kg/day, animals of either sex treated with 150 or 75 mg/kg/day or surviving recovery animals.

Organ Weights. Males treated with 300 mg/kg/day showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. Females from this treatment group also showed a statistically significant increase in relative liver weight. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg/day.

Histopathology

The following treatment-related changes were observed:

Salivary Gland:A higher incidence of lower grades of secretory activity of the submaxillary salivary glands was seen in relation to treatment for animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day and also for females treated with 75 mg/kg/day. Treatment-related changes in secretory activity of the salivary glands can be regarded as adaptive.
The condition was observed to have regressed among Recovery 300 mg/kg/day males following an additional fourteen days without treatment, but a marginal residual effect on secretion remained for Recovery 300 mg/kg/day females.

Bone Marrow:A higher incidence of higher grades of severity of adipose infiltration of the bone marrow, indicative of reduced cellularity, was seen for males only treated with 300 mg/kg/day. A similar effect was not seen for females or for males from any other treatment level. The condition had regressed among Recovery 300 mg/kg/day males after completion of the recovery period.


Conclusion

The oral administration of the test material to rats by gavage, at dose levels of 75, 150 and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated at 300 and 150 mg/kg/day and in females treated with 75 mg/kg/day. A 'No Observed Effect Level' (NOEL) for systemic toxicity for females has therefore not been established; however, the 'No Observed Effect Level' (NOEL) for systemic toxicity for males was considered to be 75 mg/kg/day.

The effects detected at 150 mg/kg/day and in 75 mg/kg/day females were confined to adaptive changes in the salivary glands. In the absence of any associated changes these effects were considered not to represent an adverse health effect. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 150 mg/kg/day.

No treatment-related effects were detected in the reproductive parameters measured. The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 300 mg/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: appr. 12 wks
- Weight at study initiation: (P) Males: 372-450 g; Females: 205-272 g
- Fasting period before study: no
- Housing: in groups of five
- Diet (e.g. ad libitum): ad-libitum
- Water (e.g. ad libitum): ad-libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55+/- 15 %
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 42 (recovery + 14 days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 15, 30, 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): --
- Purity: distilled
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of N-Butylpyridinium Chloride in the test material formulations was determined by high performance liquid chromatography (HPLC).
The test material formulations were diluted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Procedure:
HPLC: Agilent Technologies 1050/1200, incorporating autosampler and workstation
Column: Sonoma 5µ C 18 (250 x 4.6 mm id)
Mobile phase: 0.1% orthophosphoric acid: methanol (90:10 v/v)
Flow-rate: 1 ml/min
UV detector wavelength 259 nm
Injection volume: 25 µL
Retention time: - 3.3 mins
Duration of treatment / exposure:
42 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Dose level Male Female
-------------------------------------------------
0 10 + 5 (recovery) 10 + 5 (recovery)
75 10 10
150 10 10
300 10 + 5 (recovery) 10 + 5 (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose level Male Female
-------------------------------------------------
0 10 + 5 (recovery) 10 + 5 (recovery)
75 10 10
150 10 10
300 10 + 5 (recovery) 10 + 5 (recovery)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before dosing, soon after dosing, and one hour after dosing and then daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing, soon after dosing, and one hour after dosing and then daily

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 and then weekly (males) and Days 0, 7, 14 and 20 post coilum(females)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
During the maturation period, weekly food consumption was recorded for each cage of
non-recovery adults. This was continued for males after the mating phase. For females
showing evidence of mating, food consumption was recorded for the periods covering
Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded
on Days 1 and 4 post partum. Food consumptions were performed weekly for each cage
of recovery adults throughout the study period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Water intake was observed daily by visual inspection of water bottles for any overt
changes.
- Time schedule for examinations:

OTHER:

- Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals
were observed for signs of functionallbehavioural toxicity. Functional performance tests
were also perfonmed on five selected males and females from each non-recovery dose
level, prior to termination , together with an assessment of sensory reactivity to various
stimuli.

- Behavioural Assessment:
Detailed individual clinical observations were performed for each animal using a
purpose-built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymatation
Hyper/Hypothermia
Skin co lour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation
This test was developed from the methods used by Irwin (1968) and Moser et./ (1988).

-Functional Performance Tests
-Sensory Reactivity
-Laboratory Investigations (Hematology, Blood Chemistry, Urinalysis)
-Reproduction Screening (Mating, Pregnancy and Parturition, Litter data, Physical Development)
-Pathology (Organ weights, Histopathology)





Sacrifice and pathology:
yes
Statistics:
Standard statistical methods have been applied for data processing.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One non-recovery female treated with 300 mg/kg/day was killed in extremis on Day 37. Two further non-recovery females from this treatment group were found dead on Days 14 and 39 and one 300 mg/kg/day recovery male was also found dead and cannibalised on Day 56. One control female was killed in extremis on Day 37. There were no further unscheduled deaths during the study.

Animals of either sex treated with 300 and 150 mg/kg/day and females treated with 75 mg/kg/day showed episodes of increased salivation immediately post or one hour post dosing. Hunched posture and lethargy were evident in one 300 mg/kg/day male on Day 21 and one female from this treatment group also showed increased lachrymation on Day 38. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated from all treatment groups throughout the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically significant effects were detected in bodyweight
development.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No effects

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating. There were no treatment-related effects on mating or conception rates for
animals of either sex treated with 300, 150 or 75 mg/kg/day.
Fertility. There were no treatment-related effects on conception rates.
Gestation Length. There were no differences in gestation lengths. The distribution for
treated females was comparable to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males treated with 300 mg/kg/day showed a statistically significant
increase in liver weight both absolute and relative to terminal bodyweight. Females from
this treatment group also showed a statistically significant increase in relative liver
weight. No such effects were detected in animals of either sex treated with 150 or
75 mg/kg/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related macroscopic abnormalities were detected for surviving
animals of either sex treated with 300 mg/kg/day, animals of either sex treated with
150 or 75 mg/kg/day or surviving recovery animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
SALIVARY GLAND: A higher incidence of lower grades of secretory activity of the
submaxillary salivary glands was seen in relation to treatment for animals of either sex
treated with 300 mg/kg/day or at 150 mg/kg/day and also for females treated with
75 mg/kg/day. Treatment-related changes in secretory activity of the salivary glands can
be regarded as adaptive.
The condition was observed to have regressed among Recovery 300 mg/kg/day males
fo llowing an additional fourteen days without treatment, but a marginal residual effect on
secretion remained for Recovery 300 mg/kg/day females.

BONE MARROW: A higher incidence of higher grades of severity of adipose infiltration
of the bone marrow, indicative of reduced cellularity, was seen for males only treated
with 300 mg/kg/day. A similar effect was not seen for females or for males from any other
treatment level.
The condition had regressed among Recovery 300 mg/kg/day males after completion of
the recovery period.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels

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Key result
Dose descriptor:
NOEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs

Target system / organ toxicity

Critical effects observed:
no
Lowest effective dose / conc.:
75 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
salivary glands

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to rats by gavage, at dose levels of 75, 150 and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated at 300 and 150 mg/kg/day and in females treated with 75 mg/kg/day. A 'No Observed Effect Level' (NOEL) for systemic toxicity for females has therefore not been established however the 'No Observed Effect Level' (NOEL) for systemic toxicity for males was considered to be 75 mg/kg/day. The effects detected at 150 mg/kg/day and in 75 mg/kg/day females were confined to adaptive changes in the salivary glands. In the absence of any associated changes these effects were considered not to represent an adverse health effect. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 150 mg/kg/day. No treatment-related effects were detected in the reproductive parameters measured. The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 300 mg/kg/day.
Executive summary:

Study Design

The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996).

Methods

The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SO) IGS BR strain rats. for up to fortyfive consecutive days (including a two week maturation phase. pairing. gestation and early lactation for females). at dose levels of 75. 150 and 300 mglkg/day. A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Two recovery groups, each of five males and five females. were treated with the high dose (300 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioural assessments, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition. haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Pairing of animals within each non-recovery dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each nonrecovery dose group after the completion of the mating phase, and for five selected parental females from each non-recovery dose group on Day 4 post partum.

Non-recovery males were terminated on Day 43, followed by the termination of all surviving non-recovery females and offspring on Day 5 post parium. All non-recovery animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Recovery animals were treated according to the dose group continuously up to the point of sacrifice of the non-recovery males at which time the treatment was discontinued. After fourteen days without treatment, the recovery males and females were subjected to a gross necropsy examination of selected tissues.

Results

Adult Responses:

Mortality

One non-recovery female treated with 300 mg/kg/day was killed in extremis on Day 37. Two further non-recovery females from this treatment group were found dead on Days 14 and 39 and one 300 mg/kg/day recovery male was also found dead and cannibalised on Day 56. One control female was killed in extremis on Day 37. There were no further unscheduled deaths during the study.

Clinical Signs

Animals of either sex treated with 300 and 150 mg/kg/day and females treated with 75 mg/kg/day showed episodes of increased salivation immediately post or one hour post dosing. Hunched posture and lethargy were evident in one 300 mg/kg/day male on Day 21 and one female from this treatment group also showed increased lachrymation on Day 38. Instances of generalised staining around the mouth, snout and eyes were detected for animals of either sex treated from all treatment groups throughout the study.

Behavioural Assessment

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Bodyweights

No toxicologically significant effects were detected in bodyweight development.

Food Consumption and Food Efficiency

No adverse effects in food consumption or food efficiency were detected.

Water Consumptions

Males treated with 300 and 150 mg/kg/day showed a substantial increase in water consumption from initiation during Week 3 until the end of the treatment period. Recovery 300 mg/kg/day animals continued to show an increase in water consumption during the first week of the recovery period. Females treated with 300 mg/kg/day showed an increase in water consumption during gestation. No such effects were detected in females treated with 150 mg/kg/day or animals of either sex treated with 75 mg/kg/day.

Haematology

Males treated with 300 mg/kg/day showed a statistically significant increase in haemoglobin and haematocrit at the Day 42 evaluations. No such effects were detected in females treated with 300 mg/kgl/day or animals of either sex treated with 150 or 75 mg/kg/day.

Blood Chemistry

Males treated with 300 mg/kg/day showed a statistically significant increase in aspartate aminotransferase at both the Day 14 and Day 42 evaluations. Females from this treatment group also showed an increase in aspartate aminotransferase at the Day 4 post parium evaluations. Following fourteen days without treatment, recovery 300 mg/kg/day males continued to show an increase in aspartate aminotransferase. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg/day.

Urinalysis

There were no treatment-related effects on urinalytical values measured.

Reproductive Performance

Mating.There were no treatment-related effects on mating or conception rates for animals of either sex treated with 300, 150 or 75 mg/kg/day.

Fertility.There were no treatment-related effects on conception rates.

Gestation Length.There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses

Offspring Litter Size and Viability.Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were comparable to controls.

Offspring Growth and Development.
Offspring bodyweight gain and litter weights at birth and subsequently on Days 1 and 4 post partum were comparable to controls.

Litter observations.No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology

Necropsy.No treatment-related macroscopic abnormalities were detected for surviving animals of either sex treated with 300 mg/kg/day, animals of either sex treated with 150 or 75 mg/kg/day or surviving recovery animals.

Organ Weights. Males treated with 300 mg/kg/day showed a statistically significant increase in liver weight both absolute and relative to terminal bodyweight. Females from this treatment group also showed a statistically significant increase in relative liver weight. No such effects were detected in animals of either sex treated with 150 or 75 mg/kg/day.

Histopathology

The following treatment-related changes were observed:

Salivary Gland:A higher incidence of lower grades of secretory activity of the submaxillary salivary glands was seen in relation to treatment for animals of either sex treated with 300 mg/kg/day or at 150 mg/kg/day and also for females treated with 75 mg/kg/day. Treatment-related changes in secretory activity of the salivary glands can be regarded as adaptive.
The condition was observed to have regressed among Recovery 300 mg/kg/day males following an additional fourteen days without treatment, but a marginal residual effect on secretion remained for Recovery 300 mg/kg/day females.

Bone Marrow:A higher incidence of higher grades of severity of adipose infiltration of the bone marrow, indicative of reduced cellularity, was seen for males only treated with 300 mg/kg/day. A similar effect was not seen for females or for males from any other treatment level. The condition had regressed among Recovery 300 mg/kg/day males after completion of the recovery period.


Conclusion

The oral administration of the test material to rats by gavage, at dose levels of 75, 150 and 300 mg/kg/day resulted in treatment-related effects in animals of either sex treated at 300 and 150 mg/kg/day and in females treated with 75 mg/kg/day. A 'No Observed Effect Level' (NOEL) for systemic toxicity for females has therefore not been established; however, the 'No Observed Effect Level' (NOEL) for systemic toxicity for males was considered to be 75 mg/kg/day.

The effects detected at 150 mg/kg/day and in 75 mg/kg/day females were confined to adaptive changes in the salivary glands. In the absence of any associated changes these effects were considered not to represent an adverse health effect. The 'No Observed Adverse Effect Level' (NOAEL) was therefore considered to be 150 mg/kg/day.

No treatment-related effects were detected in the reproductive parameters measured. The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 300 mg/kg/day.