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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Jan 2009 - Nov 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was performed for the ultimate degradation product of the test material in aqueous environment. The study design of this GLP compliant assay was fully compliant with OECD TG 422.

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)
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 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
Details on mating procedure:
- M/F ratio per cage:
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 / concentrations
Remarks:
Doses / Concentrations:
75, 150, 300 mg/kg/day
Basis:
analytical conc.
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

Examinations

Parental animals: Observations and examinations:
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)





Oestrous cyclicity (parental animals):
yes
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight
Litter observations:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post parium
iii) Sex of offspring on Day 1 and 4 post parium
iv) Clinical condition of offspring from birth to Day 5 post parium
v) Individual offspring and litter weights on Day 1 and 4 post partum
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulation gland
Colon
Duodenum
Epididymides
Eyes·
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries
Pancreas
Pituitary
Prostate
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Skin (hind limb)
Spinal cord (cervical, mid·thoracic and
lumbar)
Spleen
Stomach
Thyroid/parathyroid
Trachea
Testes.
Thymus
Urinary bladder
Uterus/Cervix
Vagina
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 5 of age.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Thyroid
Liver Uterus

Statistics:
Standard statistical methods have been applied for data processing.
Reproductive indices:
Mating performance
Litter Responses
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: systemic effects
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: systemic efects
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
other: reproductive parameters

Overall reproductive toxicity

Reproductive effects observed:
not specified

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:

This 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).

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 forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 75, 150 and 300 mg/kg/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 treat-ment for a further fourteen days.

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.