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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:
experimental study
Adequacy of study:
key study
Study period:
from 10 september 2008 to 15 september 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study without any deviations
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (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 animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd.; Laboratory Animal Services; Wölferstrasse 4; 4414 Füllinsdorf / Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 288 to 334 g; Females: 185 to 209 g
- Fasting period before study: no
- Housing: individually (except during paring period) in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Randomization: yes, Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Diet: Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no. 77/07).
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: yes 6 days, under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): There was a 12-hour fluorescent light / 12-hour dark cycle with music during the light period.

IN-LIFE DATES: From: september 10th, 2008 to january 31th 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.
PMP was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle (highly purified water) was added (w/v).
Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle
was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation
- Proof of pregnancy: sperm in vaginal smear or copulation plug, referred to as day 0 of post coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days).
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories.
The formulations were found to be in the acceptable range of nominal concentration, homogeneous and stable when kept 4 hours and 7 days at
room temperature.
Duration of treatment / exposure:
Paramethoxyphenol was administered to male rats for at least 28 days and to
female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1
generation reached day 4 post partum.
Frequency of treatment:
daily
Details on study schedule:
- pre pairing period: 14 days
- pairing period: 14 days maximum (all females mated during the first pre pairing period)
- gestation: 21 days
- treatment ends: on day 4 post partum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150 and 300 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 per sex and per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: as agreed with the Sponsor, based on results of a range finding study (Harlan B96131, non GLP), using the dose
levels of 100, 300 and 500 mg/kg bw/d. No reprotoxic effects were seen in all treatment groups. For general toxicity,
a NOEL of 100 mg/kg bw/d and a LOEL of 300 mg/kg bw/d were identified.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside
the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions,
and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling
as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Males: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14 and weekly after pairing periods
Females: Pre-pairing period days 1-4, 4-8, 8-11 and 11-14; gestation days 0-7, 7-14 and 14-21
post coitum, and days 1-4 post partum.
No food consumption was recorded during the pairing period.

OTHER: Viability / Mortality: twice daily

FUNCTIONAL OBSERVATION BATTERY (FOB):
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were
performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily
dose administration. Animals were observed for the following:
a) Cage-side observations: unusual body movements, abnormal behavior and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing, responsiveness to sharp noise, paw pinch, gait
evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability
(Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151
(FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and
the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATION
Blood samples were obtained on the day before or on the day of the scheduled necropsy from 5 males (randomly selected) from each group.
Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all
animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water
ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HEMATOLOGY
The following hematology parameters were determined:
Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean
corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, total Leukocyte count,
Differential leukocyte count: Platelet count
Coagulation: Prothrombin time (= Thromoplastin time), Activated partial Thromoplastin time.

CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides,
Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Creatine kinase, Sodium,
Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin and Albumin/Globulin ratio

Oestrous cyclicity (parental animals):
During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular
estrus cycles.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, sperm staging
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, sex ratio

GROSS EXAMINATION OF DEAD PUPS:
yes, macroscopically
Postmortem examinations (parental animals):
SACRIFICE
Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
Dams were sacrificed on day 5 post partum.
Dams which birth did not occur on the expected date (day 21 post coitum) were sacrificed on day 26 post coitum.

GROSS NECROPSY
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to
establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral
phosphate buffered 4% formaldehyde solution (except for testis and epididymides in Boin’s
fixative).
All animals were sacrificed by an injection of sodium pentobarbital. All P generation animals
were exsanguinated.
Dead pups, except those excessively cannibalized, were examined macroscopically.
All parent animals and pups were examined macroscopically for any structural changes, either at
the scheduled necropsy .
For the parent animals, special attention was directed at the organs of the reproductive system.
The number of implantation sites and corpora lutea per uterus horn were recorded for all dams
with litters. The uteri of non-pregnant females and of females not littering were placed in a
solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites,
live/dead embryos, early and late embryonic deaths.

ORGAN WEIGHTS
The testes and epididymides of all parental males were weighed as pairs.
In addition, from 5 males and females killed at the end of the study which were selected from
each group, the following organs were trimmed from any adherent tissue, as appropriate, and
their wet weight taken:
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the
control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions.
tissues collected:
Prostate
Seminal vesicles with coagulating gland
Testes (in Bouin’s fixative)
Epididymides (in Bouin’s fixative)
Ovaries
Gross lesions
Brain
Spinal chord
Small and large intestines (incl. Peyer’s patches)
Stomach
Liver
Kidneys
Adrenals
Spleen
Heart
Thymus
Thyroids, and parathyroids if possible
Trachea and lungs (preserved by inhalation with fixative and then immersion)
Uterus (with vagina)
Urinary bladder
Lymph nodes (mesenterial, mandibular)
Peripheral nerve (sciatic)
Bone marrow

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial
cell structure.
Histological examination of ovaries was carried out on female that did not give birth. In addition,
microscopic examination of the reproductive organs of infertile male was made.
Postmortem examinations (offspring):
SACRIFICE
Pups were sacrificed on day 4 post partum.

GROSS NECROPSY
dead pups were examined macroscopically only.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and
reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance
estimate was applied if the variables could be assumed to follow a normal distribution
for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopical findings.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time,
post-implantation losses, mean litter size, pup sex ratios and viability indices.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated for offsrping: mean litter size, pup sex ratios and viability
indices.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

The reproduction and breeding data are detailed here, the other parameters are detailed in the endpoint of repeated exposure, for the same study.

- Mating Performance and Fertility
All females mated within the first pairing period.
The median and mean precoital times were unaffected by treatment with the test item.
Due to one female in group 2 that was not pregnant, the fertility index and conception rate were
100% in groups 1, 3 and 4 and 90% in group 2.

- Duration of Gestation
The mean duration of gestation was unaffected by exposure to the test item and within the range
of the historical control data. Mean duration of gestation was 21.3, 21.4, 21.6 and 21.4 days, in
order of ascending dose level.

- Corpora Lutea Count
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (15.4,
15.1, 14.8 and 15.4 in order of ascending dose level) and within the range of the historical
control data and therefore gave no indication of a test item-related effect.

- Implantation Rate and Post-implantation Loss
The mean number of implantations per dam was similar in all groups and within the range of the
historical control data.
In group 3, total post-implantation loss was statistically significantly increased, while mean
incidence of post-implantation loss as a percentage of total implantations was also increased but
not statistically significant. This was due to the higher post implantation loss which occurred in
one dam which was noted to have difficulty in delivery. In absence of any dosedependency
and since the value was within the range of the historical control data, this was not
considered to be a test item-related effect.

- Litter Size at First Litter Check
In group 3, due to increased incidence of post implantation loss in one dam (noted to have
difficulty in delivery) and to the higher postnatal loss at first litter check in one other dam, the birth
index (number of pups born alive/ number of implantations) resulted to be statistically
significantly lower (85.7%). Since no effects were observed in group 4 and the value was within
the range of the historical control data, this was considered to be incidental.

- Sperm Staging
Under the conditions of this study, the test item did not reveal effects on the completeness of
stages or cell populations. There was no indication for maturation arrest, re-absorption of sperms
or any other degenerative type.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: mating performance, duration of gestation, corpora lutea count, implantation rate and litter size

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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

- Postnatal Loss Days 0 - 4 Post Partum
No test item-related effects were noted in the postnatal loss until day 4 postpartum.
Mean postnatal loss was 0.4, 0.0, 0.6 and 0.3% in group 1, 2, 3 and 4, respectively.
Correspondingly, the viability index was 97.2, 100.0, 95.0 and 97.4% which was within the
range of the historical control data.

- External Examination at First Litter Check and during Lactation
No abnormal findings were noted at first litter check or during the first 4 days post partum.
In group 3, one male pup was noted to have no milk in the stomach.

- Sex Ratios
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test
item. In group 4, the statistically significantly lower incidence of female pups at first litter check
was considered to be incidental since it was within the range of the historical control data.
The proportion of males on day 4 post partum was 44, 50, 46 and 54%, in order of ascending
dose level.

- Body Weights to Day 4 Post Partum
Mean pup weights on day 1 post partum were unaffected by treatment with the test item.
During the lactation period mean pup weight development was not affected by the treatment with
the test item.

- Macroscopical Findings
No test item-related findings were noted at macroscopic examination of F1 pups.
In group 4, autolysis was noted in one male pup found dead on day 1 post partum.
In group 3, increased size was noted for one female pup which was found dead at first litter
check. Autolysis was observed in four pups of same litter found dead on day 1 post partum and
no milk in the stomach was observed in male pup found dead at first litter check.
No macroscopical findings were observed in group 2.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 300 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: offspring viability, body weight, gross pathology effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the clinical signs, reduced body weight, body weight gain and reduced food consumption observed
at 300 mg/kg bw/day, a general NOAEL was established at 150 mg/kg bw/day.
The relevant reproduction parameters were not affected by the treatment with the test item.
Therefore the NOAEL for reproduction/developmental toxicity was considered to be greater than 300 mg/kg bw/day, for
mating performance, duration of gestation, corpora lutea count, implantation rate and litter size.
Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage

administration of the test item Paramethoxyphenol RNCAS 150-76-5 to rats over approximately

28 days. Paramethoxyphenol was administered in highly purified water as vehicle at dosages of

50, 150, and 300 mg/kg bw/day, and controls received the vehicle only. Paramethoxyphenol

was administered to male rats for at least 28 days and to female rats for 14 days prior to

pairing, through the pairing and gestation periods until the F1 generation reached day 4 post

partum.

Administrations at 300 mg/kg bw/day caused a reduction of the activity and signs of discomfort

in all animals for the entire treatment period. Ruffled fur and difficulty in delivery occurred in

three females. Food consumption, body weight and body weight gain were reduced for the whole

pre-pairing period in males and females. All these effects were attributed to the treatment.

At 150 mg/kg bw/day, the transient lower food consumption noted in males and females was not

considered to be adverse since it affected marginally the body weight or body weight gain and

recovered afterwards. At this dose level, two females were noted to have ruffled fur

and difficulty in delivery. Same females were noted to have higher post-implantation loss (one female)

and higher postnatal loss at first litter check (the other one). Although postimplantation

and postnatal losses were statistically significantly higher there was no dose dependency.

Thus, these effects were not considered to be adverse.

The parameters investigated during the functional observational battery did not indicate any test

item-related effect.

The relevant reproduction parameters (number of corpora lutea, implantation rate, postimplantation

loss, number of fetuses and post-natal loss) were not affected by the treatment with

the test item. The statistically significant increase of post implantation loss and decrease in birth

index observed in two females at 150 mg/kg bw/day were not dose dependent.

The assessment of hematology and clinical biochemistry data and histopathology examination

did not reveal any test item-related effects. The organ weights were also not affected by the

treatment with the test item.

Based on the clinical signs, reduced body weight, body weight gain and reduced food

consumption observed at 300 mg/kg bw/day, a general NOAEL was established at 150 mg/kg bw/day.

The relevant reproduction parameters were not affected by the treatment with the test item,

except at 150 mg/kg bw/day, where an increase of post implantation loss and a decrease of birth

index were noted (only for two females). However, these effects were not dose dependent and

within the range of historical control data.

Therefore the NOAEL for reproduction/developmental toxicity was considered to be greater than 300 mg/kg body weight/day.