Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
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
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
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
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
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.
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
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
- 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.
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
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Study following the OECD 422 guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

One study is referenced under the section toxicity to the reproduction, but it is a combined repeated dose toxicity study with the reproduction/developmental toxicity in the rat.

It was rated as Klimisch 1, GLP study without deviation, and it was chosen as key study.

Paramethoxyphenol was administered orally 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.

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 (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, 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.

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

Therefore, based on this study, PMP is not expected to have impact on reproduction or fertility.

Moreover, in several carcinogenic studies no data on reproductive organs have been reported, suggesting certainly that these organs were not affected. Therefore, taking into account all these elements, a 2 generation study does not seem to be needed. 


Short description of key information:
Based on the OECD 422 test guideline study, the NOAEL for reproduction/developmental toxicity was considered to be greater than 300 mg/kg body weight/day, the higher tested dose.

Justification for selection of Effect on fertility via oral route:
GLP study, OECD 422 compliant

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 03 december 2012 to June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 414 compliant
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Number: 96 female rats were received at CiToxLAB France between 07 and 21 December 2012.
- Strain and sanitary status: Sprague-Dawley, Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Breeder: Charles River Laboratories Italia, Calco, Italy.
- Age/Weight: at the beginning of the treatment period, the females were 10-11 weeks old and had a mean body weight of 265 g (range: 224 g to 323 g). The females were sexually mature and primigravid.
- Housing: The animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2, 48 cm x 26.5 cm x 21 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen because it is preferable for pregnant animals.
Each cage contained an object for the environmental enrichment of the animals (rat hut).
The cages were placed in numerical order on the racks.
- Food and water: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 2537604 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly. The diet formula is presented in Appendix 3.
The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation: the animals were acclimated to the conditions of the study for a period of 4 or 5 days before the beginning of the treatment period (arrival of the females on day 1 or 2 p.c.).
- Allocation to study: before the beginning of the treatment period, the animals were allocated to the groups, according to a stratification procedure based on body weight recorded on day 2 p.c., to ensure comparatively similar mean body weights of the groups.

- Identification: each animal was individually identified by an ear tattoo (unique CiToxLAB France identity number).

ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: about 12 cycles/hour of filtered, non-recycled air.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Dose formulation preparation
The test item was administered as a solution in the vehicle.
Before preparing the dose formulations, the test item was ground, using a mortar and pestle. Then, the required quantities were mixed progressively with the vehicle in order to obtain the desired concentration.
After addition of the vehicle, the dose formulations were kept under magnetic stirring for at least 30 minutes to ensure effective solubilization of the test item.
The test item dose formulations were prepared for up to 11 days, stored at room temperature and protected from light and delivered in brown flasks.

VEHICLE
- Lot/batch no. (if required): The vehicle was drinking water treated by reverse osmosis using ELIX 5 (Millipore SA).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item in the dose formulations have been quantified by a validated analytical method.
The validation of the analytical method was conducted in CiToxLAB France/Study No. 39417 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the corresponding validation report.
Details on mating procedure:
- Mating: the females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).
Duration of treatment / exposure:
once daily, from days 6 to 20 p.c., inclusive, to time-mated female
Frequency of treatment:
Daily
Duration of test:
Two months and a half
Remarks:
Doses / Concentrations:
100, 200 and 400 mg/kg/d bw
Basis:
nominal conc.
No. of animals per sex per dose:
24 mated female per group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected by the Sponsor, following the results of a previous study: Combined repeated dose toxicity with the reproduction/developmental toxicity screening test, according to the OECD 422 guideline (report B96142, Sept. 2009).
Maternal examinations:
Morbidity and mortality
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

Clinical signs
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

Body weight
The body weight of each female was recorded on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

Food consumption
The quantity of food consumed by each female was recorded for the following intervals:
- days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21
- Macroscopic post-mortem examination of the principal thoracic and abdominal organs.
- Other organs examined: ovaries, uterus
Any macroscopic lesions observed were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative).
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with discernible digits.

Uterine horns without visible implantation sites were immersed in an aqueous solution of ammonium sulphide (Salewski, 1964) to reveal the presence of uterine scars, which were counted.

A gross evaluation of placentas was also undertaken.

Body weight of fetuses
The body weight of each live fetus was recorded.

Sex of fetuses
The sex of each fetus was determined at the time of hysterectomy.
The sex of fetuses was determined by visual assessment of anogenital distance and was confirmed by examination of sexual organs at detailed dissection of the soft tissues or at evisceration.
Fetal examinations:
Fetal examination was conducted for all litters where the female had at least one live fetus.

External examination
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.

Soft tissue examination
As soon as possible after sacrifice, approximately half of the live fetuses in each litter were subjected to a detailed dissection of the soft tissues, which included the observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated and were fixed with Harrison's fluid for examination of the structures of the head.

Skeletal examination
The remaining live fetuses per litter was eviscerated and then fixed with ethyl alcohol.
A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
PREGNANCY STATUS: At termination on day 21 p.c., there were 22, 23, 21 and 18 rats with live fetuses and 2, 1, 3 and 4 non-pregnant females in the groups treated at 0, 100, 200 and 400 mg/kg/day, respectively. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day.

MORTALITY: There were no unscheduled deaths.

CLINICAL SIGNS (Table 1): Ptyalism was considered to be related to the treatment with the test item, but of minor toxicological significance.
Hypoactivity and half-closed eyes were observed from 100 mg/kg/day, piloerection, locomotory difficulties and sedation from 200 mg/kg/day and, round back and tonic contraction at 400 mg/kg/day. All these clinical signs were recorded with dose-related increased incidence/severity and were considered to be test item treatment-related from 100 mg/kg/day and of toxicological signifance from 200 mg/kg/day.

BODY WEIGHT: At 200 mg/kg/day and when compared with controls, there were a statistically significant minimal decrease in mean body weight (-6.7%, on day 9 p.c., p<0.05) which resulted in a statistically significant lower mean body weight gain (+3 g vs. +16 g in controls, p<0.001).
At 400 mg/kg/day and when compared with control values, there was a marked mean body weight loss (-19 g) over the period of days 6 to 9 p.c., thereafter mean body weight gain returned toward control values and decreased from day 15 to 21 p.c.. During all the treatment period, there were statistically significant decreases in mean body weight (up to -15.8% on day 21 p.c., p<0.01).
All these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day.

FOOD CONSUMPTION: At 100 mg/kg/day and when compared with controls, there were a statistically significant but minimal decrease in mean food consumption at initiation of the treatment period (-11.1% on days 6-9 p.c., p<0.05) which returned to control values thereafter.
At 200 mg/kg/day and when compared with controls, there were a statistically significant and marked decrease in mean food consumption at initiation of the treatment period (-33.3% on days 6-9 p.c., p<0.05) which increased thereafter but remained lower than controls at the end of the treatment period (-9.7% on days 18-21 p.c., p<0.01).
At 400 mg/kg/day and when compared with controls, there were a statistically significant and severe decrease in mean food consumption at initiation of the treatment period (-63% on days 6-9 p.c., p<0.001) which never returned to control values (-19.4% on days 18-21 p.c., p<0.01).
All these findings were considered to be test item treatment-related from 100 mg/kg/day and toxicologically significant from 200 mg/kg/day.

MACROSCOPIC post-mortem EXAMINATION: At 400 mg/kg/day, one female (A25766) showed a dilatation of pelvis (kidneys) and ureter. These findings are commonly observed in this species and strain; therefore, a test item treatment-related effect was considered unlikely.

NET BODY WEIGHT CHANGE: When compared with controls, there was a statistically significant decrease in mean uterus weight at 400 mg/kg/day (-18.7%, p<0.05). Taking into account the amplitudes of the changes, this finding was considered to be toxicologically significant.
When compared with controls, there were statistically significant decreases in carcass weight at 200 and 400 mg/kg/day (-7.7%, p<0.25 and -14.8%, p<0.01; respectively), resulting in dose related decreased mean body weight changes.
Taking into account the amplitude of the changes, these findings were considered to be test item treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day.

HYSTERECTOMY DATA (Table 2): At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean implantation loss (19.9% vs. 2.2, p<0.01) corresponding mainly to early resorptions (females A25765 and A25766 had 100% post-implantation losses and female A25770 had 66.7% post-implantation loss) which resulted in decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05). All these findings were considered to be toxicologically significant.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FETAL BODY WEIGHT AND SEX RATIO: When compared with controls, there was a dose-related decrease in mean fetal body weight from 100 mg/kg/day which resulted in a statistically significant difference at 400 mg/kg/day (-15.9%, p<0.01). At 200 mg/kg/day, mean fetal body weight was below the lower limit of the Historical Control Data (5.35 g vs. 5.5 g, respectively). However, taking into account the amplitude of the changes this finding was considered to be toxicologically significant only at 400 mg/kg/day.
There were no effects on sex ratio (mean percentage of male fetuses).

EXTERNAL EXAMINATION (Tables 3 and 4): Malrotated limb, curled tail and domed head were not previously recorded in contemporaneous Historical Control Data. When compared with the control group or to the Historical Control Data, the increased litter and fetal incidences of external variations at 400 mg/kg/day were considered to be related to the test item treatment.
Exencephaly was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

SOFT TISSUE EXAMINATION (Tables 5 and 6): When compared with the control group or Historical Control Data, there were no test item treatment-related soft tissues variations.
Misshapen cerebrum was recorded at comparable incidence in the control and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

CARTILAGE AND SKELETAL EXAMINATION (Tables 7 and 8): In control, 100 and 200 mg/kg/day group, there were no toxicologically significant findings at cartilage examination.
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in fused cartilage of cervical vertebra(e) [11.1 (3.5 *) vs. 0.0 (0.0) in terms of litter (fetal) incidences, p<0.05) and two fetuses from the same litter (A25763) with fused cartilage of ribs. Taking into account the findings described below, these observations were considered to be related to the test item treatment.
Forepaw(s) with unossified distal phalanx, unossified 1st metatarsal, hindpaw(s) with unossified distal phalanx, were observed at comparable litter and/or fetal incidences across groups, therefore a test item treatment-related effect was considered unlikely for these observations.
However, when compared with the control group or to the Historical Control Data, the increased litter and fetal incidences in all other skeletal variations at 400 mg/kg/day were considered to represent significant ossifications delay and to be related to the test item treatment.

Absent lumbar vertebra(e) was recorded at comparable incidence in the control, 100 and 400 mg/kg/day groups. Therefore, a test item treatment-related effect was considered unlikely for this finding.
However, all other malformations with increased litter and fetal incidences at 400 mg/kg/day were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

DISTRIBUTION OF FETAL MALFORMATION (table 9): Taking into account both litter and fetal incidences, there was a marked increase of malformations at 400 mg/kg/day.
Abnormalities:
not specified
Developmental effects observed:
not specified

MATERNAL DATA

Table 1: Maternal clinical signs

Dose-level (mg/kg/day)

0

100

200

400

Piloerection

/

/

9

(from day 7 up to 20 p.c.)

10

(from day 7 up to 20 p.c.)

Round back

/

/

/

1

(from day 7 to 9 p.c.)

Tonic contraction

/

/

/

13

(from day 6 up to 13 p.c.)

Hypoactivity

/

14

(from day 6 up to 20 p.c.)

24

(from day 6 up to 20 p.c.)

23

(from day 6 up to 20 p.c.)

Locomotory difficulties

/

/

9

(from day 6 up to 18 p.c.)

17

(from day 6 up to 20 p.c.)

Sedation

/

/

11

(from day 6 up to 20 p.c.)

19

(from day 6 up to 13 p.c.)

Ptyalism

/

1

(from day 18 up
to 20 p.c.)

3

(from day 15 up to 18 p.c.)

3

(from day 16 up to 20 p.c.)

Half-closed eyes

 

12

(from day 6 up
 to 20 p.c.)

24

(from day 6 up to 20 p.c.)

23

(from day 6 up to 20 p.c.)

Number of affected animals

0/24

15/24

24/24

24/24

( ): in brackets, days of first and last occurrence of the clinical signs.

Table 2: Hysterectomy data

Dose-level (mg/kg/day)

0

100

200

400

HCD
[min.-max.]

Number of females with live fetuses

22

23

21

18

150(a)

Mean number ofcorpora luteaper animal

15.6

15.4

16.4

14.8

14.0 - 15.5

Mean number of implantations per animal

14.1

13.6

14.8

13.2

12.8 - 14.0

Mean pre-implantation loss (%)

9.6

12.8

10.1

11.4

7.2 - 13.9

Mean number of live fetuses per litter

13.8

13.3

14.1

11.1*

12.0 - 13.2

Mean number of early resorptions

0.3

0.3

0.6

2.0**

/

Mean number of late resorptions

0

0

0

0.1

/

Mean post-implantation loss (%)

2.2

1.6

4.7

19.9**

2.0 - 8.7

Statistical significance:*: p<0.05, **: p<0.01.

HCD: Historical Control Data.

(a): control data collected from seven studies covering a period ranging from February 2008 to March 2012.

/: not listed.

FETUS DATA

Table 3: Litter (L) and Fetal (F) incidences of external variations

Dose-level (mg/kg/day)

0

100

200

400

HCD

Dams with live fetuses, n

22

23

21

18

143(a)

Fetuses examined, n

303

307

296

222

1813

Malrotated limb, L(F) %

 

 

 

5.6 (0.5)

-

Curled tail, L(F) %

 

 

 

5.6 (0.5)

-

Domed head, L(F) %

 

 

 

5.6 (0.5)

-

Litters affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

2 (11.1)

3 (2.1)

Fetus affected, n (%)

0 (0.0)

0 (0.0)

0 (0.0)

3 (1.4)

16 (0.9)

Table 4: Litter (L) and Fetal (F) incidences of external malformations

Dose-level (mg/kg/day)

0

100

200

400

HCD

Dams with live fetuses, n

22

23

21

18

143(a)

Fetuses examined, n

303

307

296

222

1813

Tera (b), L(F) %

 

 

 

5.6 (0.5)

-

Anasarca, L(F) %

 

 

 

5.6 (0.5)

-

Cleft lip, L(F) %

 

 

 

11.1 (2.3*)

-

Cleft palate, L(F) %

 

 

 

11.1 (2.3*)

-

Omphalocele, L(F) %

 

 

 

11.1 (3.2**)

-

Short trunk, L(F) %

 

 

 

5.6 (0.5)

-

Short tail, L(F) %

 

 

 

5.6 (0.5)

-

Exencephaly, L(F) %

4.5 (0.3)

 

 

5.6 (1.4)

-

Litters affected, n (%)

1 (4.5)

0 (0.0)

0 (0.0)

4 (22.2)

1 (0.7)

Fetus affected, n (%)

1 (0.3)

0 (0.0)

0 (0.0)

10** (4.5)

1 (0.1)

Statistical significance:*: p<0.05, **: p<0.01.

HCD: Historical Control Data.

(a): control data collected from seven studies covering a period ranging from February 2008 to March 2012.

/: not listed.

Conclusions:
Up to 200 mg/kg/day, the test item elicited no developmental toxicity in a context of moderate to marked maternal toxicity as demonstrated by adverse clinical signs (hypoactivity, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption.

At 400 mg/kg these findings were more severe and associated with increased post-implantation losses (mainly as a consequence of increased early resorptions). At this dose level only and in such a context, developmental delays (reduced affecting fetal body weight and ossification associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded.
Executive summary:

The objective of this prenatal development toxicity study (2013) was to evaluate the potential toxic effects of the test item, Paramethoxyphenol, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive).

Three groups of 24 time-mated Sprague-Dawley rats were administered the test item, Paramethoxyphenol, once daily from day 6 to day 20 p.c., by gavage at dosages of 100, 200 or 400 mg/kg/day. An additional group of 24 time-mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as the control group. A dose volume of 12 mL/kg/day was used.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recordedat designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

Up to 200 mg/kg/day, the test item elicited no developmental toxicity in a context of moderate to marked maternal toxicity as demonstrated by adverse clinical signs (hypoactivity, locomotory difficulties and sedation)and, decreased mean body weight, mean body weight change and/or mean food consumption.

At 400 mg/kg these findings were more severe and associated with increased post-implantation losses (mainly as a consequence of increased early resorptions). At this dose level only and in such a context, developmental delays (reduced affecting fetal body weight and ossification associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse clinical signs (hypoactivity, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption from 200 mg/kg/day and increased post-implantation losses (mainly as a consequence of increased early resorptions) at 400 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 200 mg/kg/day, based on increases in variations (external and skeletal) and malformations (external, soft tissues and skeletal) at 400 mg/kg/day in a context of marked maternal toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study following the OECD 414 guideline.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Two studies were quoted in this section, of reliability 3:

- In the first one (Kavlock RJ, 1990), SD female rats were administered by gavage a dose of PMP once, at GD11. The offsprings were observed until day 6 post partum. No developmental effects were observed.

- the second one ( Oglesby LA, 1992) used SD rat embryos that were cultured with PMP.

These two studies were not standard protocols and did not permit to conclude about the possible developmental toxicity of PMP. That's why the testing proposal for this endpoint was a study according to OECD guideline n° 414. The summary of the study is the following:

The objective of this prenatal development toxicity study (2013) was to evaluate the potential toxic effects of Paramethoxyphenol (PMP), on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum (p.c.), inclusive).

Three groups of 24 time-mated Sprague-Dawley rats were administered PMP, once daily from day 6 to day 20 p.c., by gavage at dosages of 100, 200 or 400 mg/kg/day. An additional group of 24 time-mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as the control group. A dose volume of 12 mL/kg/day was used.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 p.c., females were sacrificed and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and/or skeletal (bones + cartilage) abnormalities.

Up to 200 mg/kg/day, PMP elicited no developmental toxicity in a context of moderate to marked maternal toxicity as demonstrated by adverse clinical signs (hypoactivity, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption.

At 400 mg/kg these findings were more severe and associated with increased post-implantation losses (mainly as a consequence of increased early resorptions). At this dose level only and in such a context,developmental delays (reduced affecting fetal body weight and ossification associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 100 mg/kg/day based on adverse clinical signs (hypoactivity, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption from 200 mg/kg/day and increased post-implantation losses (mainly as a consequence of increased early resorptions) at 400 mg/kg/day,

.         the NOAEL for embryo-fetal development was considered to be 200 mg/kg/day, based on increases in variations (external and skeletal) and malformations (external, soft tissues and skeletal) at 400 mg/kg/day in a context of marked maternal toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
GLP study OECD 414 compliant.

Justification for classification or non-classification

After the results observed in the OECD 414 study, the paramethoxyphenol should be classified as Reprotoxic Category 2 H361d (suspected of damaging the unborn child), according to the CLP 1272/2008 regulation.

This classification is based on increases in variations (external and skeletal) and malformations (external, soft tissues and skeletal) observed at 400 mg/kg/day in a contexte of marked maternal toxicity. An increase of post-implantation losses (mainly as a consequence of increased early resorptions) was also observed at 400 mg/kg/day,