Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two reprotoxic studies with reliability 1 (Klimisch cotation) are available: an OECD 422 study (Harlan, 2009) and an OECD 443 study (CitoxLab, 2019b).

Based on the OECD 422 study no effect on reproduction was observed on HanRcc: WIST(SPF) rats up to the dose level of 300 mg/kg/day. The relevant reproduction parameters were not affected by the substance Paramethoxyphenol.

Based on the OECD 443 study and when compared with controls or historical Control Data, there were no test item dose-related effect on Sprague-Dawley rats regarding effects on mating (mating index), mating behavior (time taken to mate) and fertility (fertility index). There were no test item treatment-related significant effects on mean duration of gestation, number of females with live born, percentage of pre- and post-implantation loss, pup live birth index, and pup sex ratio.

These two studies showed that the substance paramethoxyphenol has no impact on reproduction parameters and on fertility.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
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
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 dose dependency
and since the value was within the range of the historical control data, this was not
considered to be a test item-related effect and this effect was considered as incidental finding.

- 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, within the range of historical control data and thus were considered as incidental findings.

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.

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2017 to 13 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection : OECD 422 study on rats and OECD 414 study on rats
- Exclusion of extension of Cohort 1B
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration : oral route (gavage). The oral route was selected since it is a route of administration which is recommended by the regulatory authorities for this type of study.
- Choice of vehicule: water
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Mequinol (paramethoxyphenol)
- EC N°: 205-769-8
- CAS N°: 150-76-5
- Description: White flakes
- Purity: higher than 99%.



Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study and the Sprague-Dawley strain was selected since background data from previous studies are available at Citoxlab France.
Strain and sanitary status: Sprague-Dawley (RjHan: SD).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS :
- Source: Janvier, Le Genest-Saint-Isle, France.
- Number: 194 rats (97 males and 97 females) were received at Citoxlab France on 19 September 2017 and designated as F0 animals.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P generation: at the beginning of the treatment period, males were approximately 6 weeks old and females were approximately 5 weeks old.
- Weight at study initiation: P generation: at the beginning of the treatment period, males had a mean body weight of 276 g (range: 246 g to 306 g) and females had a mean body weight of 185 g (range: 155 g to 208 g).
- Fasting period before study: no
- Housing: The P generation animals were group housed in Tecniplast 2154 cages (48 x 26.5 x 21 cm) with stainless steel lids (2/sex per cage) except 2 weeks before mating and during mating, gestation and lactation (individually housed). The F1 animals were housed in Tecniplast 2154 cages (48 x 26.5 x 21 cm) with stainless steel lids (4/sex per cage for cohort 1A animals, 5/sex per cage for cohort 2A and 2/sex per cage for cohort 1B animals) Cages containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen since it is preferable for pregnant animals, littering and lactating females in order to not jeopardize gestation, littering and lactation phases, and to avoid aggressive behavior around mating in males.Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
Each cage contained two objects (nylabone and rat hut or cocoon) for the environmental enrichment of the animals. The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos 64619633, 45722325 and 21024582 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: males and females were acclimated to the study conditions for a period of 10 days before treatment. Two supplementary animals (one male and one female) were acclimated to permit selection and/or replacement of individuals.
- Allocation to group: before the beginning of the treatment period, the required number of animals (96 males and 96 females) was selected according to body weight and clinical condition and allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar (i.e. ± 20% of the global mean values among the groups).
- Identification: the P generation animals were individually identified by an implanted microchip (unique Citoxlab France identity number). The pups of the P generation were identified by a toe tattoo on Day 1 post-partum (p.p.). The pups selected to constitute the F1 cohorts were identified by an implanted microchip at weaning.
- Contaminant analyses: The batches of diet, sawdust and wood shavings were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which may be expected to interfere with or prejudice the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C,
- Humidity (%): 50 ± 20%,
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air,
- Photoperiod (hrs dark / hrs light): 12h/12h.
- The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.

IN-LIFE DATES:
P generation:
- males: From 19 september 2017 (arrival) to 07 February 2018
- females: From 19 september 2017 (arrival) to 03 February 2018 (last female).

F1 generation:
- Cohort 1A: From 22 January (first animal) to 10 April 2018 (last animal).
- Cohort 1B: From 22 January (first animal) to 13 April 2018 (last animal).
- Cohort 2A: From 22 January (first animal) to 22 March 2018 (last animal).
- Cohort 2B: From 22 January (first animal) to 28 January 2018 (last animal).
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Vehicle: drinking water treated with reverse osmosis
- Concentration in vehicle: 0, 3.3, 8.3 and 20.8 mg/mL (corresponds to 0, 40, 100 and 250 mg/kg/day)
- For P generation a constant dosage-volume of 12 mL/kg/day was used.


Control animals (group 1) received the vehicle only.
The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the dosing procedure.
The control dose formulation was stirred just before administration and the test item dose formulations for at least 15 minutes before administration.
The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.
The treatment was performed once daily at approximately the same time, each day (7 days a week) and the days of developmental landmark testing, when animals were treated after the test had finished (this could be in the afternoon).


Details on mating procedure:
- M/F ratio per cage: One female was placed with one male. Females were paired with males from the same dose level group.
- Length of cohabitation: Each female was placed with the same male until mating occurred or 14 days had elapsed.
For scheduling purposes (to avoid excessive numbers of animal at necropsy), the mating period started over 4 different days (1st day: first six surviving males/females, 2nd day: following up to six surviving males/females and unmated pairs, 3rd day: following up to five or six surviving males/females and unmated pairs, 4th day: last following surviving males/females and unmated pairs).
- Proof of pregnancy: Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c.
- When mating had not occurred after 2 weeks, the animals were separated without further opportunity for mating.
- The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical technique: High Performance Liquid Chromatography with UV detection (HPLC/UV).

Principle and validation of the method: Analytical method developed and validated at Citoxlab France (Citoxlab France/Study No. 39417 VAA, 04-Jan-2013).
Checked parameters, acceptance criteria and obtained results are described in the validation report.

Determination of test item concentrations in dose formulations: Eight dosages: three times on P-premating, one in each P-mating and P-gestation, and three on F1-postweaning periods. A sample was taken from control and test item dose formulations and analyzed using the validated method.

Acceptance criterion: measured concentration = nominal concentration ± 10%
Duration of treatment / exposure:
For males and females P generation: Starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, F1 generation was also exposed to graduated doses of the test item while being assigned to cohorts of animals for reproductive/developmental toxicity or developmental neurotoxicity testing. See details in the field 'Details on study schedule' below.


Frequency of treatment:
Daily.
Details on study schedule:
- Selection of parents from F1 generation when pups were 22 days of age (see detail below).
- Age at mating of the mated animals in the study: arround 15 weeks for females / arround 16 weeks for males.

P-generation:
Sexually-mature male and female rats (parental (P) generation) were exposed to graduated doses of the test item. The dose formulations were administered daily according to the following schedule:
in the P males (at least 18 weeks of treatment):
- 10 weeks before mating,
- during the mating period (up to 2 weeks),
- until euthanasia (after weaning of the pups, i.e. at least 6 weeks after mating),
in the P females (at least 16 to 18 weeks o of treatment):
- 10 weeks before mating,
- during the mating period (up to 2 weeks),
- during gestation,
- during lactation until Day 21 p.p. (or Day 22 p.p. sacrifice after hematology, blood chemistry and urinalysis on Day 23 p.p. for the selected females),
- until euthanasia for females with no evidence of mating or no delivery (24-26 days after the last day of the mating period).

Day 1 corresponds to the first day of the treatment period.
Day 0 p.c. (post-coitum) corresponds to the day of confirmed mating (detection of a vaginal plug or sperm cells in a vagina smear).
Day 0 p.p. (post-partum) corresponds to the day on which parturition occurs.
Day 1 p.p. corresponds to the day on which parturition is completed.

At weaning, pups were selected and assiged to cohorts of animals. The F1 offspring received further treatment with the test item from weaning to adulthood or sacrifice. The dose formulations were administered daily according to the following schedule:

Cohort 1A:
In the males and females:
- from weaning (Day 22 p.p.),
- until euthanasia (animals euthanized from Days 92 to 96 p.p.).

Cohort 1B:
In the males and females:
- from weaning (day 22 p.p.) for approximately 11 weeks,
- until euthanasia (after necropsy of cohort 1A pending no alteration of estrous cycle or sperm parameters, and no macroscopic finding after reproductive organs examination were observed).

Cohort 2A:
In the males and females:
- from weaning (day 22 p.p.)
- until euthanasia (animals euthanized after completion of behaviotal testing: Days 75 to 78 p.p.).

Cohort 2B:
There was no direct dosing in cohort 2B animals (euthanized on Day 22 p.p.).

Administration:
The dose formulations were administered by gavage using a plastic syringe fitted with a plastic gavage tube (juvenile gavage tubes from Days 22 to 33 p.p. and adult gavage tubes from Day 34 p.p.), once a day, at approximately the same time of day.
The quantity of the dose formulations administered to each animal was adjusted according to the most recently recorded body weight.


Control animals (group 1) received the vehicle only.

The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the dosing procedure.
The control dose formulation was stirred just before administration and the test item dose formulations for at least 15 minutes before administration.
The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
P generation: 24 rats per sex and per dose.
F1 generation: 20 rats per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor, based on the results of two studies:
1) an OECD 422 (combined repeated dose toxicity study with the reproduction/developmental toxicity screening test) performed in 2009 (RCC/Study No. B96142) in which MEQUINOL (PARAMETHOXYPHENOL) was administered by gavage to Wistar Han rats (RCC Wist, SPF) at 0, 50, 150 and 300 mg/kg/day. Maternal effects were observed at 300 mg/kg/day (NOAEL = 150 mg/kg/day). No reprotoxic effects were observed in this study,
2) an OECD 414 (prenatal development toxicity study) performed in 2013 (Citoxlab France/Study No. 39419 RSR) in which MEQUINOL (PARAMETHOXYPHENOL) was administered by gavage to time-mated female Sprague-Dawley rats, once daily (from Days 6 to 20 p.c. inclusive), at 0, 100, 200 or 400 mg/kg/day. Based on this study a NOAEL of 100 mg/kg/day has been identified for maternal effects (hypoactivity, locomotor difficulties and sedation, decreased mean body weight and mean body weight change). The NOAEL for embryo-fetal development was considered to be 200 mg/kg/day, based on increased variations (external and skeletal) and malformations (external, soft tissues and skeletal) at 400 mg/kg/day in a context of marked maternal toxicity.
Therefore, 250 mg/kg/day was selected as the high-dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 2.5-fold interval (i.e. 40 and 100 mg/kg/day).

- Rationale for animal assignment (if not random):Pups were selected randomly.
- Fasting period before blood sampling for clinical biochemistry: Prior to blood sampling and urine collection at termination, the first 10/sex surviving P males, the first 10/sex littering P females and the first 10/sex surviving cohort 1A animals from each group were deprived of food for an overnight period of at least 14 hours.

- Other (details):
In the assay, sexually-mature male and female rats (parental (P) generation) were exposed to graduated doses of the test item starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for:
- a) reproductive/developmental toxicity testing: cohort 1A and cohort 1B (without mating to produce a F2 generation),
- b) developmental neurotoxicity testing: cohorts 2A and 2B.

The F1 offspring received further treatment with the test item from weaning to adulthood or sacrifice. Clinical observations and pathology examinations were performed on all animals (Parental and F1 generation) for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive systems and the health, growth, development and function of the offspring.

On Day 22 p.p., pups from all available litters (up to 20 litters per group) were assigned to four cohorts of animals, as follows:
- cohorts 1A and 1B = reproductive/developmental toxicity testing,
- cohorts 2A and 2B = developmental neurotoxicity testing.

Pups were selected randomly, with the exception that obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not included, as they were unlikely to be representative of the treatment group.
The pups selected to constitute the F1 cohorts (with exception of pups allocated to cohort 2B sacrificed on Day 22 p.p.) were identified by an implanted microchip (unique Citoxlab France identity number) at weaning.
Day 22 p.p. was designated Day 1 of the F1 generation.
Parental animals: Observations and examinations:
For P and F1 generations after weaning:


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
P0: From arrival, each animal was observed once a day as part as routine examinations. From the start of the treatment period each animal was observed at least twice a day (before and after treatment), at approximately the same time of day, for the recording of clinical signs.
Detailed clinical examinations were performed on all animals once a week until the end of the study.
Observations included (but were not limited to) changes in the 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, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.
F1: Each animal was observed at least twice a day (before and after treatment), at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations:
P0: The body weight of each male was recorded on the first day of treatment (Study Day 1), then at least once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Study Day 1), then at least once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p. Animals were weighed at euthanasia as normal procedure before pathology examination.
F1: The body weight of each animal (cohorts 1A, 1B and 2A) was recorded on the first day of treatment (Day 1), then at least once a week until euthanasia.

FOOD CONSUMPTION: Yes
- Time schedule:
P0: The quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period and at least once a week after the mating period until euthanasia.
The quantity of food consumed by each female was measured at least once a week from the first day of treatment until the start of the mating period, during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14-21 p.p..
During the mating period, food consumption was not measured for males or females.
F1: The quantity of food consumed by each animal was measured at least once a week from the first day of treatment (cohorts 1A, 1B and 2A) until euthanasia


OTHER:
- Medical care: On a few occasion several animal from all groups received topical application of Cothivet® (essential oils) and/or Antisept® (chlorhexidine). These healing and antiseptic sprays were used to treat wounds/scabs .

- Morbidity and mortality:
P0: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.
Attention was paid to humane end-points defined in Citoxlab France quality document LI SES 0004. End points specific to the species used in the study were available in the animal facility.
Animals showing signs of poor clinical condition were weighed and humanely euthanized.
F1: Each animal was checked for mortality and morbidity at least twice a day during the treatment period, including weekends and public holidays.
Attention was paid to humane end-points defined in Citoxlab France quality document LI SES 0004.
End-points specific to the species used in the study were available in the animal facility.

- Sexual development
All male animals were observed each day from Day 40 p.p. (i.e. Day 19 of F1 generation), until cleavage of the balanopreputial groove (preputial separation) was observed (when not observed before, until
euthanasia, for cohorts 1A and 2A animals). All female animals were observed each day from Day 28 p.p. (i.e. Day 7 of F1 generation), until vaginal opening was observed (when not observed before, until euthanasia, for cohorts 1A and 1B animals)
The body weight of each animal was recorded on the day of balanopreputial separation or vaginal opening.

Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during the last 2 weeks of the premating period,
- during the mating period, until the females were mated or the mating period had ended,
A fresh vaginal lavage was done on the day of sacrifice (premature or scheduled).
Sperm parameters (parental animals):
Full seminology investigations were performed on all surviving P males from the control- and high-dose groups (groups 1 and 4). Since sperm motility was assessed immediately after sampling, this was also performed on all surviving P males from the low- and intermediate-dose groups (groups 2 and 3). Slides for the assessment of sperm morphology were prepared for all surviving P males from the low- and intermediate-dose groups (groups 2 and 3). The left testis and left caudal epididymis of all surviving P males from the low- and intermediate-dose groups (groups 2 and 3) were sampled and stored at -20°C pending possible sperm count evaluation. The left testis and left caudal epididymis of all surviving cohort 1A males from the low- and intermediate dose groups (groups 2 and 3) were used for testicular sperm head cells count (including calculation of the daily testicular sperm production rate) and epididymal sperm cells count.

Epididymal sperm:
Under deep anesthesia and after epididymis weight, sperm from the cauda of the left epididymis was sampled for motility and morphology investigations. Then, animals were euthanized. The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently frozen at -20°C for further investigation.

Sperm motility:
Epididymal sperm motility: Sperm was evaluated on a slide, after appropriate dilution. The number of motile and non-motile spermatozoa was evaluated under a microscope using a 40-fold magnification from a sample of 200 spermatozoa. Results were expressed as the percentage of motile and non-motile spermatozoa.

Sperm morphology:
Epididymal sperm morphology: Sperm morphology was determined from a smear, after eosin staining, counting 200 spermatozoa per slide. Results were expressed as the percentage of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Sperm count:
Epididymal sperm count: After thawing, the left cauda epididymis was weighed, minced and homogenized using a Polytron in a saline-triton solution. An aliquot of the suspension was sampled and the number of spermatozoa was counted in a Malassez cell. Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm:
After thawing, the left testis was weighed and grounded. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a Neubauer cell. Results were expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10; Blazak et al., 1993).

Macroscopic examination:
Special attention was paid to the males reproductive organs
Testes and epididymides were preserved in Modified Davidson's Fixative.

Testicular staging (groups 1 and 4): a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, and allowed detection of retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

Epididymis (groups 1 and 4): examination included the caput, corpus and cauda and allowed detection of leukocyte infiltration, change in prevalence of cell types, aberrant cell types, phagocytosis of sperm, etc.

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes.
- On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible five males and five females per litter. Whenever necessary, partial adjustment (for example six males and four females) was permitted.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter size: the total litter size was recorded on Day 1 p.p. The litters were observed daily in order to note the number of live, dead and cannibalized pups.
Sex of pups: the sex of each pup was recorded on Day 1 P.P.
Clinical signs: The pups were observed daily for clinical signs, abnormal behavior and external abnormalities (including oral cavity and orifices). The first clinical examination (on Day 1 p.p.) included: gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion), qualitative assessment of body temperature, activity and reaction to handling.
Body weight: The weight of each live pup was recorded on Days 1, 4, 7, 14 and 21 of p.p.
Pup development: The following physical development measurements were performed in pups of each litter: anogenital distance (AGD) on Day 4 p.p. (all pups before culling), number of nipples and of areolae in male pups: on Day 13 p.p. The AGD was normalized to the cube root of body weight recorded on Day 4 p.p.


GROSS EXAMINATION OF DEAD PUPS:
Yes. Pups prematurely euthanized because of dying mother were deeply anesthetized by an intraperitoneal injection of Euthasol. A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead (except cannibalized) and prematurely euthanized pups. Special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). Macroscopic lesions were preserved in appropriate fixative for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Cohort 2A: neurobehavioral tests: On Day 22 p.p., 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least one pup; randomly selected) were selected for neurobehavioral testing followed by neurohistopathology assessment as adults.
Auditory startle test: Cohort 2A animals were subjected to an auditory startle test on Day 24 p.p. (± 1 day) using a computerized shuttle box system to determine the amplitude and time to response.
Reactivity to manipulation or to different stimuli (Functional Observation Battery): Cohort 2A animals were observed once or twice between Days 65 and 72 p.p., in the cage, in the hand and in the standard arena. Where possible the same observer evaluated the animals in a given test. The following parameters were assessed and graded:
in the cage: "touch escape" or ease of removal from the cage,
in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
in the standard arena (2-minute recording): grooming, palpebral closure, defecation and urination counts, tremors, twitches, convulsions (tonic and clonic), gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia, hypotonia.
In addition, the following parameters, reflexes and responses were recorded: pupil reflex, auditory startle reflex, forelimb grip strength and visual stimulus.

Motor activity:
Cohort 2A animals were subjected to motor activity testing at the same time as the clinical examination for reactivity to manipulation or to different stimuli using an automated infra-red sensor equipment recording individual animal activity over a 60-minute period.
The following parameters were reported: horizontal movements and vertical movements.

Neuropathology assessment:
Cohort 2A: Cohort 2A animals were terminated after behavioural testing, with brain weight recorded and full neurohistopathology for purposes of neurotoxicity assessment.
Cohort 2B: On Day 22 p.p., 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least one pup; randomly selected) were selected for neurohistopathology assessment at weaning (Day 22 p.p.) (see below).

Neurohistopathology:
Neurohistopathology was performed for all high-dose and control animals of each sex following completion of neurobehavioral testing (between Days 75 and 78 p.p. for cohort 2A animals and on Day 22 p.p. for cohort 2B animals). Animals from cohort 2A were perfusion-fixed (the vascular system was flushed by saline, followed by a perfusion with 10% neutral buffered formalin).
Multiple sections were examined from the brain to allow examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. The eyes (retina and optic nerve) and samples of peripheral nerve, muscle and spinal cord were also examined.

Organ weight measurements: The brain and the pituitary gland of all animals were fixed in formalin after opening the brain cavity, but with the brain supported by the skull base; after fixation the brain and the pituitary gland were removed and weighed with a precision of 0.001 g after study termination. Weighing was performed approximately 72 hours ± 24 hours after necropsy to prevent physical damage of the soft tissue during weighing.
Tissue preservation: All tissue samples were processed to paraffin wax blocks embedded. The brain tissue from all dose-groups was processed to blocks at the same time to avoid shrinkage artefacts associated with prolonged storage in fixative.

Histopathology examinations
Control and high-dose groups were processed to slides. As test-item treatment-related changes were found in the high-dose group, examination of tissues from mid- and low- dose group were performed:
- Brain morphometric measurements:
. Cohort 2A: measurements of sensory cortex thickness in low- and mid-dose males (measurements L3 L2+6).
. Cohort 2B: measurements of motor cortex thickness in low- and mid-dose females (measurements L3 L1+5).

Morphometric (quantitative) evaluations were performed on representative areas of the brain (homologous sections carefully selected based on reliable microscopic landmarks) and included linear and/or areal measurements of specific brain regions.
Each specific brain region was embedded in different paraffin blocks: one per brain level area (cerebral cortex, medulla/pons and cerebellar sections).

At least three step sections (200 µm apart) were made at each level in order to select the most homologous and representative section for the specific brain area to be evaluated. Non-homologous sections were not used for evaluation. A minimum of 6 (up to 10) brains/sex was evaluated for the control and high-dose groups.
The appropriate relative dimensions of the cerebral cortex, hippocampus and cerebellum were assessed in each examined brain.
All aspects of the preparation of tissue samples, from tissue fixation, through the dissection of tissue samples, tissue processing, and staining of slides, were employed a counterbalanced design, such that each batch contained representative samples from each dose group where possible.




Postmortem examinations (parental animals):
SACRIFICE:
On completion of the treatment period, all surviving P animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination:
P males: after weaning of most of the F1 progeny (the left epididymis was sampled after anesthesia and before euthanasia, see above section 'Sperm parameters').
P females: on Days 22-23 p.p.,
P females which did not deliver: on Days 24-26 p.c. (after body weight recording to check for a possible unnoticed delivery),
P female with no evidence of mating: 25 days after the end of the mating period since no delivery occurred,
P females with total litter loss: as appropriate.


P and F1 generation adults:

Organ weights: The body weight of each animal euthanized as scheduled was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table (see table below in the section 'Any other information on materials and methods incl tables') were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Macroscopic post-mortem examination: A complete macroscopic post-mortem examination was performed on all P. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were recorded for females euthanized on Days 22-23 p.p. for P generation.

Preservation of tissues: The tissues specified in Tissue Procedure Table were preserved in 10% buffered formalin (see table below in the section 'Any other information on materials and methods incl tables') (except for the eyes with optic nerves, testes and epididymides which were preserved in Modified Davidson's Fixative). Ovaries (all groups from P generations) were fixed for at most 96 hours in formalin before being embedded in paraffin wax.

Preparation of histological slides: All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS and for the right ovaries which were stained by PCNA immunohistochemistry). Five step-sections (100 µm apart in the middle third) were made for the right ovary. This tissue processing was performed at Citoxlab France.

Microscopic examination: A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from animals of the control and high-dose groups (groups 1 and 4) euthanized at the end of the treatment period (see table in the section 'Any other information on materials and methods incl. tables)',
- reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal oestrous cycles and from any male with abnormalities at sperm analysis, to investigate possible causes (low- and mid-dose groups),
- all macroscopic lesions of all groups (from P), including those from prematurely euthanized animals.

Testicular staging (groups 1 and 4): a detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle, and allowed detection of retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

Epididymis (groups 1 and 4): examination included the caput, corpus and cauda and allowed detection of leukocyte infiltration, change in prevalence of cell types, aberrant cell types, phagocytosis of sperm, etc.

Right ovary (groups 1 and 4): a detailed and careful microscopic examination was made of five step sections of the right ovary of each P and cohort 1A female, with enumeration of the total number of primordial follicles on PCNA-stained slides.

Based upon the results of the microscopic examination of the high-dose group and after the agreement of the Sponsor, other tissues of the low- and intermediate-dose groups have been examined:
- Microscopic examination on hematoxylin-eosin stained slides :
. forestomach from low- and mid-dose groups of P-Generation (males and females) animals,
. bone marrow (sternum) from low- and mid-dose groups of P-Generation (males and females) animals.
- Enumeration of the total number of primordial follicles on PCNA-stained slides:



Postmortem examinations (offspring):
SACRIFICE
- F1 pups were euthanized by an intraperitoneal injection of sodium pentobarbital followed by exsanguination:
pups whose mother died: as soon as possible,
pups not selected on Day 4 p.p.: on Day 4 p.p.,
pups not selected at weaning: on Day 22 p.p.

These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
A complete macroscopic post-mortem examination was performed on all F1 animals (including F1 pups culled on Day 4 p.p. and not selected F1 pups on Day 22 p.p.). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
Preservation of tissues: The tissues specified in Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes with optic nerves, testes and epididymides which were preserved in Modified Davidson's Fixative, see § Study plan adherence).
Ovaries of F1 generations were fixed for at most 96 hours in formalin before being embedded in paraffin wax.


For more details, see 'Tissue Procedure Table' in the section below 'Any Other information on materials and methods incl. tables'.
Statistics:
Body weight, food consumption and reproductive data:
Data were compared by one-way variance analysis and Dunnett's test (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).

Organ weight:
PathData software was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).

Auditory startle reflex test and number of primary follicles:
Statistical analysis was performed using the SAS Enterprise Guide software.
To compare the auditory startle reflex response (categorical data using positive or negative values) between treatment groups, a Fisher's exact test was used. If a global statistically significant difference is observed between treatment groups, multiple Fisher's exact tests were performed to compare the auditory startle reflex responses of each test item-treated group with that of the control group.

No-genital distance, locomotor activity, number of nipples and areolae, time of preputial separation/vaginal opening, time to first estrous after vaginal opening/patency, seminology, hematology, blood biochemistry, urinalysis, thyroid hormones and post-implantation loss:
Citox software was used to perform the statistical analyses.
Reproductive indices:
The following parameters were calculated: Post-implantation loss, mating index, fertility index and gestation index as indicated below:

- post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________x 100
Number of implantation sites

- mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

- fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

- gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females


Parturition:
Females were allowed to litter normally and rear their progeny until weaning. Any sign of a difficult or prolonged parturition (dystocia) was recorded. The day of completed parturition was designated Day 1 p.p. The length of gestation was calculated. Any abnormalities in nesting behavior or nursing performance were recorded.




Offspring viability indices:
The following parameters were calculated: live birth index, vialibility index on Day 4 p.p., lactation index as indicated below:

- live birth index:
Number of live pups on Day 1 p.p.
_____________________ x 100
Number of delivered pups

- viability index on Day 4 p.p.:
Number of surviving pups on Day 4 p.p. (before culling)
_______________________________________ x 100
Number of delivered pups

- lactation index:
Number of surviving pups on Day 21 p.p.
________________________________________ x 100
Number of surviving pups on Day 4 p.p. (after culling)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
At 250 mg/kg/day, half-closed eyes was recorded in three male rats during the Days 70-120 of the treatment period and was considered to be test item treatment-related. Similar to the observed hypoactivity, this finding was transitory (observed for 1 to 2 hours) after dosing and was not correlated with macroscopic or microscopic (including brain histomorphology examination) findings. Staggering gate was observed in one single male sporadically on Days 105-109, therefore a test item treatment relationship was considered unlikely.
From 100 mg/kg/day, hypoactivity was observed in all treated male rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
Ptyalism was observed from 40 mg/kg/day at increased dose-level incidences and was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.

A series of additional clinical signs were observed in males without any dose level relationship (i.e. abnormal growth of teeth, alopecia/thinning of air, chromodacryorrhea, chromorhinorrhea, enophthalmos, exophthalmos, eye opacity, lacrimation, liquid discharge (nose), reflux at dosing, scabs/wounds, thin appearance). They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Overall, in P generation males and from 100 mg/kg/day neurological clinical signs, such as hypoactivity, were observed in all animals together with half-closed eyes at 250 mg/kg/day. Even if these clinical signs were transitory after dosing, with low incidence for half-closed eyes and not correlated with macroscopic and microscopic findings, these clinical signs have to be taken into consideration since they were also observed in females (see below) and also in all cohorts (see below appropriate sections). Furthermore, they are consistent with a mild effect on the central nervous system (CNS), which is a common trait of many quinones (Topping et al., 2007).


Females:
At 250 mg/kg/day, half-closed eyes was recorded in four female rats on the Days 70-73 of the treatment period and was considered to be test item treatment-related. This finding was observed at low incidence, transitory (observed for 1 to 2 hours) after dosing and was not correlated with macroscopic or microscopic (including brain histomorphology examination) findings. Staggering gate/tonic seizures were observed in only one female on Day 99, therefore a test item treatment relationship was considered unlikely.
From 100 mg/kg/day, hypoactivity was observed in almost all treated female rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
Ptyalism was observed from 40 mg/kg/day at increased dose level incidences and was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.

A series of additional clinical signs were observed in females without any dose level relationship (i.e. abnormal growth of teeth (cut regularly), hunched posture, piloerection, loud breathing, alopecia/thinning of hair, chromodacryorrhea, chromorhinorrhea, increase in size (mammary gland, urogenital region), liquid discharge (nose), pallor (extremities, eye(s)), reflux at dosing, scabs/wound, soiled (urogenital area, neck), thin appearance and/or vaginal discharge). They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Overall, in P generation females and from 100 mg/kg/day neurological clinical signs were observed in almost all rats together with half-closed eyes at 250 mg/kg/day. Even if these clinical signs were transitory after dosing, with low incidence for half-closed eyes and not correlated with macroscopic and microscopic findings these clinical signs have to be taken into consideration since these effects were seen in all males (see previous section) and also in all cohorts (see below appropriate sections). Furthermore, they are consistent with a mild effect on the central nervous system (CNS), which is a common trait of many quinones (Topping et al., 2007).

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Mortality:
mortality observed, non-treatment-related
Description (incidence):

The unscheduled deaths recorded in the P generation is the following:
M-L21921 (male, control group): with lateral recumbency on Study Day 108 was prematurely sacrificed for humane reasons. There were no associated findings at necropsy.
M-L21940 (male, 40 mg/kg/day): with a right limb increased in size on Study Day 77 was prematurely sacrificed for human reasons. At necropsy the right tibia had a red solid mass (approx 0.8 cm in diameter).
M-L21997 (male, 250 mg/kg/day): the plastic gavage tube was broken during the gavage procedure on Study Day 114 and swallowed; the male was prematurely sacrificed for human reasons. At necropsy a piece of the gavage tube was present in esophagus up to stomach.
F-L24413 (female, control group): with pallor and piloerection from Study Day 96, and apparent difficulties to deliver on Study Day 97 (eight pups alive in the litter) was prematurely sacrificed for human reasons. At necropsy, there were three dead fetuses with placenta and nine placentas in the left uterine horn .
F-L24427 (female, control group): the plastic gavage tube was broken on Study Day 54 during the gavage procedure and swallowed; the female was prematurely sacrificed for human reasons. At necropsy there was a piece of the gavage tube into the stomach.
F-L24471 (female, 100 mg/kg/day): with hunched posture, pallor of eyes and extremities, piloerection, dyspnea, vaginal discharge and dead litter on Study Day 99 was prematurely sacrificed for human reasons. At necropsy, this female had tan discoloration of lungs/bronchi, liver and kidneys, gelatinous thymus and a dead fetus in the vagina.
F-L24476 (female, 100 mg/kg/day): with hypoactivity, lateral recumbency, tonic seizures, hypotonia and dyspnea on Study Day 49 was prematurely sacrificed for human reasons. At necropsy, there was an enlarged spleen. A technical issue at gavage was considered to be the most probable cause of death.
F-L24488 (female, 250 mg/kg/day): with hunched posture, pallor of extremities, piloerection from Day 99 and, hypoactivity regularly from Day 1, and dead litter on Study Day 99 was prematurely sacrificed for human reasons. There were no findings at necropsy.
F-L24491 (female, 250 mg/kg/day): with pallor of extremities, piloerection and cold to the touch on Day 99, hypoactivity regularly from Day 1, and difficulties to deliver on Study Day 99 (two live pups and four dead pups in the litter) was prematurely sacrificed for human reasons. At necropsy, this female had the right uterine horn with nine dead fetuses with placentas and a left uterine horn with eight scars.

Decisions of sacrifice for difficulties to deliver or dead litter were taken for control and/or treated animals. These findings are frequent observations in animalsof this physiological condition and commonly recorded in this species and strain. Therefore a test item treatment-related effect was considered to be unlikely.
Overall, there were no tendencies towards test item treatment-related deaths in male or female animals from the P Generation.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Premating period:
Males:
At 250 mg/kg/day, when compared with controls, there was a lower mean body weight (down to -9% on Day 126, p<0.001; +317 g vs. +374g on Days 1 to 126, p<0.001) and a low mean body weight gain (down to +317 g vs. +374 g in control on Days 1 to 126, p<0.001). These findings were considered to be test item treatment-related and adverse (no recovery in term of body weight gain).
At 100 and 40 mg/kg/day, when compared with control, there were no effects.

Females:
At 250, 100 and 40 mg/kg/day, when compared with controls, there were no effects on mean body weight and no test item treatment-related effects on mean body weight change.

Pregnancy period (females):
At 250, 100 and 40 mg/kg/day, when compared with controls, there were no test item treatment-related effects on mean body weight and mean body weight change during the pregnancy period.

Lactation period (females):
Mean body weight: At 250, 100 and 40 mg/kg/day, when compared with controls, there were no test item treatment-related effects on mean body weight during the lactation period.
Mean body weight change: In all test item-treated groups, when compared with controls, there were lower mean body weight changes on Days 1 to 4 p.p. This finding was considered to be test item treatment-related but not adverse based on the return towards control values from Days 4 to 7 p.p.

Conclusion: effect on body weight was observed at 250 mg/kg/day and considered to be treated-related and as adverse in males.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Premating:
Males:
At 250, 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Females:
At 250 mg/kg/day, when compared with controls, there were episodes of lower mean food consumption (down to -13% on Days 36 to 43, p<0.05). This finding was considered to be test item treatment-related and adverse with moderate toxicological importance (less than 20% difference vs. controls with a return towards control values on Days 64 to 71).
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Pregnancy period (females): At 250, 100 and 40 mg/kg/day, when compared with controls, there were no effects during the pregnancy period.

Lactation period (females): At 250 mg/kg/day, when compared with controls, there was a lower mean food consumption in females (down to -18% on Days 7 to 14 p.p., p<0.01). This finding was considered to be test item treatment-related and to be adverse with moderate toxicological importance (less than 20% difference vs. controls).
At 100 and 40 mg/kg/day, when compared with controls, there were no effects during the lactation period.


Conclusion: food consumption was decreased in females during premating and during lactation. This finding was considered to be test item treatment-related and to be adverse with moderate toxicological importance.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males: at 250 mg/kg/day and when compared with controls, there were higher mean cell volume, mean cell hemoglobin, prolonged prothrombin time, and lower leucocytes, erythrocytes, hemoglobin, packed cell volume, basophils, lymphocytes and monocytes counts. Only haemoglobin was lower than the lower limit of the Historical Control Data and prothrombin time was above the upper limit of the Historical Control Data. However, there were no similar observations in females. Therefore, these findings were considered to be of non toxicological importance and to be non adverse.

In females: there were no significant effects on hematology and coagulation data.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males: at 250 mg/kg/day and when compared with controls, there were higher potassium, lower creatinine and total cholesterol concentrations. While the differences were statistically significant, the concentrations remained within the range of the Historical Control Data. In addition, there were no similar observations in females and not correlatation with histopathology examination. Therefore, these findings were considered to be of non toxicological importance and to be non adverse.

In females: there were no effects on blood biochemistry parameters.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects on urinalysis parameters.

At 40 mg/kg/day, when compared with controls, females had a lower urinary specific gravity (-1%, p<0.05). In the absence of any dose level relationship or a similar finding in males, this observation was considered fortuitous.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were noted in the forestomach from 100 mg/kg/day and in the bone marrow at 250 mg/kg/day.

. Forestomach
Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis were noted in forestomach from some males and females treated at 250 mg/kg/day (see table below). In one female treated at 250 mg/kg/day, it was associated with slight focal ulcer. In a few males and females treated at 100 mg/kg/day, minimal focal hyperkeratosis and squamous cell hyperplasia were observed.
The spectrum of lesions including squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis, and ulcer in one female, was considered to be adverse at 250 mg/kg/day in view of the severities (up to moderate) and of the nature of these lesions (subacute/chronic inflammation).
In view of the very low severity of hyperkeratosis and hyperplasia at 100 mg/kg/day and of the absence of associated ulceration, these findings were considered to be non-adverse at 100 mg/kg/day.

. Bone marrow
Increased cellularity (with increased myeloid/erythroid ratio) was seen in the bone marrow from males and females treated at 250 mg/kg/day (see table below). It was seen mostly in animals with the forestomach lesions recorded with highest grades. This finding was considered to be probably secondary to the forestomach inflammatory process and was considered to be non-adverse in view of its low magnitude and of the absence of clinical pathology correlates.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.This included, among others:
. minimal to moderate crusts and acanthosis in the skin from test item-treated males and females with poor dose-relationship common on the rats kept in laboratory conditions,
. minimal hepatocellular hypertrophy in 2/22 females treated at 250 mg/kg/day,
. minimal or marked suppurative inflammation in the kidneys from 2/22 females treated at 250 mg/kg/day.

There were no test item-related changes in the male or female reproductive organs nor in nervous system.

There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.

Refer to the attached documents (DOC 1 and DOC 1ter) in the field below 'Attached background material’.

Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones: There were no effects on mean plasma thyroid hormones (T4 and TSH) concentrations in P generation animals (males or females).

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg/day, when compared with controls, there was a higher mean number of days of diestrus (5.2 vs. 3.8, p<0.01) and a lower mean number of days of proestrus (2.4 vs. 3.6, p<0.001). This finding was considered to be test-item treatment related but not adverse as all values remained within the range of Historical Control Data.
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
At 250 mg/kg/day and when compared with controls, there were no effects on sperm analysis data in males from the P generation.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Reproductive performance:
no effects observed
Description (incidence and severity):
When compared with controls or Historical Control Data, there were no test item dose-related effects on mating (mating index), mating behavior (time taken to mate) and fertility (fertility index).
Gestation index (%) and mean duration of gestation were not affected by the test-item treatment.
There were no dose-related effects on mean percentage of post-implantation loss, live birth index and sex-ratio. Therefore a test-item treatment related effect was considered unlikely.
At 250 mg/kg/day, there was an apparent increase in mean percentage of pre-implantation loss which remained within the acceptable range of variations for this strain of rats. Therefore a test-item treatment related effect was considered unlikely.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
The main toxic effects observed in the parent generation is neurological clinical signs such as hypoactivity observed from 100 mg/kg/day and half-close eyes noted at 250 mg/kg/day showing mild impact of the substance on the central nervous system (CNS). Inflammation of the forestomach was also observed. Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis were noted in forestomach from some males and females treated at 250 mg/kg/day. In a few males and females treated at 100 mg/kg/day, minimal focal hyperkeratosis and squamous cell hyperplasia were observed.


Other results not detailed on the different sections above:
Enumeration of the total number of primordial follicles on PCNA-stained slides: There were no differences between the high-dose and the control groups, with respectively a mean total number of 5.06 and 6.96 primordial follicles per section on PCNA-stained slides.

Table: Total number of primordial follicles on PCNA-stained slides

Group 1 4
Dose level (mg/kg/day) 0 250
mean number per section 5.06 6.96
SD 5.26 3.48
min 0.00 0.20
max 18.80 12.00
See also document (DOC 1) in the field 'Attached background material'.

Other parental results are summarized in tables in the section below ‘Any other information on results incl tables’ and more complete data (tables) are provided in the field below 'below 'Attached background material’.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Clinical signs such as hypoactivity were observed from 100 mg/kg/day (CNS). Reduction of food consumption in females and decrease of body weight in males were noted at 250 mg/kg/day. Adverse forestomach inflammation was also observed in male and female parents at 250 mg/kg/day.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pups: P generation offspring refers to Days 1 to 21 p.p. pups (before weaning).
Clinical observations during lactation:
In test item-treated groups and when compared with controls, there were no pups with malformation(s) or specific test item treatment-related findings at gross external examination, qualitative assessment of body temperature and, activity and reaction to handling in Day 1 p.p. pups.
From 40 mg/kg/day, a few pups had hypoactivity. This finding was transient at 40 and 100 mg/kg/day (observed on one day) and persistent at 250 mg/kg/day (observed for 22 days). Taking into account the occurrence and reversibility of this finding, a test-item treatment-related effect was not excluded at 250 mg/kg/day.
All other findings recorded in pups during the lactation period are common observations in this species and strain of rats maintained in the experimental condition of this study.
Refer to the attached document (DOC3) in the field below 'Attached background material’.



Clinical signs for F1 cohorts:

Cohort 1A:
Males: From 100 mg/kg/day, ptyalism was recorded in all animals. This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also noted from 100 mg/kg/day (observed only 1 to 2 hours after dosing). This effect was observed in all treated cohort 1A male rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
A series of additional clinical signs were observed without any dose level relationship [i.e. alopecia, thin appearance, piloerection, abnormal growth of teeth (cut regularly), loud breathing, dyspnea, chromodacryorrhea, chromorhynorrhea, exteriorized penis and/or scabs/wounds]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.
At 40 mg/kg/day, there were no test item treatment-related findings.

Females: From 100 mg/kg/day, ptyalism was recorded in almost all animals. This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also recorded from 100 mg/kg/day and half-cllosed eyes from 250 mg/kg/day (observed only 1 to 2 hours after dosing) in almost all animals. These effects were considered to be test item treatment-related but were not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
A series of additional clinical signs were observed without any dose level relationship [i.e. alopecia, reflux at dosing, and/or scabs]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.
At 40 mg/kg/day, there were no test item treatment-related findings.

Overall, from 100 mg/kg/day neurological clinical signs (such as hypoactivity) were observed in all males and females cohort 1A. In addition, half-closed eyes were observed at 250 mg/kg/day in all female rats. Even if these clinical signs were transitory after dosing, and not correlated with macroscopic and microscopic findings these clinical signs have to be taken into consideration since these effects were seen in all the parents (males and females) and also in the othecohorts. Furthermore, they are consistent with an effect on the central nervous system (CNS), which is a common trait of many quinones (Topping et al., 2007).
Refer to the attached document (DOC4) in the field below 'Attached background material’.

Cohort 1B:
Males: From 100 mg/kg/day, ptyalism was recorded in all animals. This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also noted from 100 mg/kg/day (observed only 1 to 2 hours after dosing). This effect was observed in almost all treated cohort 1B male rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
At 40 mg/kg/day, there were no test item treatment-related findings.

A series of additional clinical signs were observed without any dose level relationship [i.e. alopecia/thinning of air, chromodacryorrhea, chromorhynorrhea, cryptorchidism (control male L22044), swelling (increased in size), reflux at dosing, loud breathing and/or scabs/wounds]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Females: From 40 mg/kg/day, ptyalism was noted (in few animals at the lower dose and in all animals at the two higher doses). This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also noted from 100 mg/kg/day (observed only 1 to 2 hours after dosing). This effect was observed in almost all treated cohort 1B female rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
At 40 mg/kg/day, there were no adverse findings.

A series of additional clinical signs were observed without any dose level relationship [i.e. hunched posture, alopecia/thinning of air, shortened tail, abnormal growth of teeth (cut regularly), chromodacryorrhea, chromorhynorrhea, reflux at dosing, tail nodosities and/or scabs/wounds]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Overall, from 100 mg/kg/day neurological clinical signs (such as hypoactivity) were observed in almost all males and females cohort 1B. In addition, half-closed eyes were observed at 250 mg/kg/day in all female rats. Even if these clinical signs were transitory after dosing, and not correlated with macroscopic and microscopic findings these clinical signs have to be taken into consideration since these effects were seen in all the parents (males and females) and also in the other cohorts. Furthermore, they are consistent with an effect on the central nervous system (CNS), which is a common trait of many quinones (Topping et al., 2007).
Refer to the attached document (DOC7) in the field below 'Attached background material’.

Cohort 2A:
Males: From 100 mg/kg/day, ptyalism was recorded in all animals. This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also noted from 100 mg/kg/day (observed only 1 to 2 hours after dosing). This effect was observed in almost all treated cohort 2A male rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
At 250 mg/kg/day, episodes of piloerection, dyspnea and staggering gait were observed only in one male after weaning (Days 1-2, i.e. Days 22-23 p.p.). While a test item treatment relationship cannot be excluded, there were considered of non-toxicological importance based on the isolated occurrence (one male for up to 2 days).
At 40 mg/kg/day, there were no test item treatment-related findings.

A series of additional clinical signs were observed without any dose level relationship [i.e. chromodacryorrhea, chromorhynorrhea, reflux at dosing and/or scabs/wounds]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Females: From 100 mg/kg/day, ptyalism was recorded in all animals. This effect was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.
Hypoactivity was also noted from 100 mg/kg/day (observed only 1 to 2 hours after dosing). This effect was observed in almost all treated cohort 2A female rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
At 250 mg/kg/day, an episode of loud breathing was observed in only one female (Day 11, i.e. Day 32 p.p.), this finding was considered to be most likely due to the gavage procedure.
A series of additional clinical signs were observed without any dose level relationship [i.e., piloerection, chromorhynorrhea, swelling (increase in size), reflux at dosing and/or scabs/wounds]. They are commonly observed findings in this species and strain of animals housed in these experimental conditions.

Overall, from 100 mg/kg/day neurological clinical signs (such as hypoactivity) were observed in almost all males and females cohort 2A. In addition, half-closed eyes were observed at 250 mg/kg/day in all female rats. Even if these clinical signs were transitory after dosing, and not correlated with macroscopic and microscopic findings these clinical signs have to be taken into consideration since these effects were seen in all the parents (males and females) and also in the other cohorts. Furthermore, they are consistent with an effect on the central nervous system (CNS), which is a common trait of many quinones (Topping et al., 2007).
Refer to the attached document (DOC11) in the field below 'Attached background material’.

Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pups: Mortality during lactation:
At 250 and 100 mg/kg/day, there were the following findings:
- Mean litter size: when compared with controls and while the differences were not statistically significant, there was an apparent dose-related decrease
with values below the lower limit of the Historical Control Data (11.7 and 10.4 vs. 11.8, at 100 and 250 mg/kg/day respectively).
- Number of dead, missing and/or cannibalized pups on Days 1-4 p.p.: when compared with controls, there was a statistically significant increase in the number of dead, missing and/or cannibalized pups. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data.

At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a consequence of lack of maternal care.

When taken together, these finding (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day.
At 40 mg/kg/day, there were no significant effects.

At 250 mg and 100 mg/kg/day, when compared with controls, there was a low number of pups surviving 4 days (with statistically significant differences: p<0.05 and p<0.001, respectively) as a consequence of the low litter sizes at birth (see above) and presence of two females with a full dead litter (L24488 and L24471 at 250 and 100 mg/kg/day, respectively). This finding was considered to be adverse and a secondary consequence of lack of maternal care at these dose levels.
At 40 mg/kg/day, there were no significant effects.
Refer to the attached document (DOC3) in the field below 'Attached background material’.

After culling on Day 4 p.p., there were no effects on mean litter sizes or lactation indexes.


Non selected pups: There was no reported mortality.

F1 cohorts:

Cohort 1A: Male L22067 (40 mg/kg/day group) with misshapen head and abnormal growth of teeth (lesion in the buccal cavity) was prematurely sacrificed
on Day 7 (Day 28 p.p.) for humane reasons.
Female L24614 (100 mg/kg/day group) was found dead on Day 22 (Day 43 p.p.) (accidental death when locking the grid to the cage box).
None of these unscheduled deaths were related to the test item treatment.
Refer to the attached document (DOC 4) in the field below 'Attached background material’.

Cohort 1B: There were no unscheduled deaths in cohort 1B males or females.

Cohort 2A: There were no unscheduled deaths in cohort 2A males or females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pups: Body weight during lactation: In test item-treated groups and when compared with controls, there were no test item treatment-related effects on pup boby weight.
At 250 mg/kg/day, when compared with controls, there was a low body weight change in female pups on Days 7 to 14 p.p. Taking into account the amplitude of the difference and the return towards control value thereafter, this finding was considered to be not adverse.
Refer to the attached document (DOC 3) in the field below 'Attached background material’.

F1 cohorts:

Cohort 1A:
Mean body weight:
Males: At 250 mg/kg/day, when compared with controls, there were episodes of lower mean body weight after weaning [down to -9% on Day 8 (Day 29 p.p.), p<0.01]. This finding was considered to be test item treatment-related and adverse.
At 100 and 40 mg/kg/day, when compared with control, there were no effects.
Females: At 250 mg/kg/day, when compared with controls, there was a lower mean body weight after weaning [down to -9% on Day 8 (Day 29 p.p.), p<0.01]. This finding was considered to be test item treatment-related and adverse.
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Mean body weight change:
At 250 mg/kg/day, in both sexes and when compared with controls, there were lower mean body weight gains after weaning [e.g. +44 g vs. +51 g in males and +36 g vs. +42 g in females, on Days 1 to 8 (Days 22 to 29 p.p.), both with p<0.01]. This finding was considered to be test item treatment-related.
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Refer to the attached document (DOC 4) in the field below 'Attached background material’.

Cohort 1B:
Males: There were no effects on mean body weights or mean body weight changes.
Females: There were no relevant effects on mean body weights and no relevant effects on mean body weight changes.

Refer to the attached document (DOC 7) in the field below 'Attached background material’.

Cohort 2A:
Mean body weight:
Males: There were no effects on mean body weight.
Females: At 250 mg/kg/day, when compared with controls, there was a lower mean body weight after weaning [down to -11% on Day 8 (Day 29 p.p.), p<0.01]. This finding was considered to be test item treatment-related but of minimal toxicological importance [based on the return towards control values from Day 22 (Day 43 p.p .)].

Mean body weight changes:
In both sexes and when compared with controls, there were lower mean body weight gains after weaning [+43 g vs. +50 g in males and +38 g vs. +44 g in females, on Days 1 to 8 (Days 22 to 29 p.p.), both with p<0.01]. ]. This finding was considered to be test item treatment-related but not adverse [based on the return towards control values from Days 8-15 (Days 29-36 p.p.)].
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Refer to the attached document (DOC 11) in the field below 'Attached background material’.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 cohorts:

Cohort 1A:
There were no significant effects on male or female mean food consumptions.
At 250 mg/kg/day and in both sexes, there were episodes of low mean food consumption after weaning [ 13% and -11% vs. controls in males and females, on Days 1 to 8 (Days 22 to 29 p.p.), respectively]. While a test item treatment relationship cannot be excluded, this finding was considered to be non adverse based on the return towards control values from Days 8 to 15 (Days 29 to 36 p.p.).

Refer to the attached document (DOC4) in the field below 'Attached background material’.

Cohort 1B:
There were no relevant test item treatment-related effects on mean food consumption.
At 250 mg/kg/day, there was an episode of low food consumption in females after weaning [-8% vs. controls on Days 1 to 8 (Days 22 to 29 p.p.)]. While a test item treatment relationship cannot be excluded (also observed in cohort 1A females), this finding was considered to be of non toxicological importance based on the return towards control values from Days 8 to 15 (Days 29 to 36 p.p.).

Refer to the attached document (DOC7) in the field below 'Attached background material’.

Cohort 2A:
There were no effects on mean food consumption in cohort 2A animals.
Refer to the attached document (DOC 11) in the field below 'Attached background material’.

Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 cohorts:

Cohort 1A:
Males: there were no effects on hematology parameters that could be related to treatment. At 40, 100 and 250 mg/kg/day, despite a few statistically significant differences when compared with controls, all values remained within the ranges of the Historical Control Data and did not correlate with any finding at histopathology.
Females: there were no effects on hematology parameters that could be related to treatment. At 250 mg/kg/day, the percentage of reticulocytes was above the upper limit of the Historical Control Data and the activated partial thromboplastin time was shortened than the control’s time. However, the magnitude of the changes were low, not dose-related, not observed in the opposite sex and/or did not correlate with any observation at histopathology; therefore these findings were considered to be of non toxicological relevance .

Refer to the attached document (DOC 4) in the field below 'Attached background material’.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 cohorts:
Cohort 1A:
In males and females, there were no effects on blood biochemistry parameters that could be related to treatment. Despite a few statistically significant differences when compared with controls, all values remained within the ranges of the Historical Control Data.
Refer to the attached document (DOC 4) in the field below 'Attached background material’.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 cohorts:
Cohort 1A:
In males and females, there were no effects on urinalysis parameters.
Refer to the attached document (DOC4) in the field below 'Attached background material’.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
F1 cohorts: The results depend on the cohorts. No effect for cohort 1A, no test item related for cohort 1B (see details below):

Cohort 1A:
Balanopreputial separation: There were no effects on the mean age of balanopreputial separation in cohort 1A males.
Vaginal opening: There were no effects on the mean age of vaginal opening in cohort 1A females.
Time to first estrous: There were no effects on the mean time from vaginal opening to first oestrous in cohort 1A females.
Estrous cycles: There were no effects on means estrous cycle parameters in cohort 1A females.

Refer to the attached document (DOC 4) in the field below 'Attached background material’.

Cohort 1B:
Balanopreputial separation: There were no effects on the mean age of balanopreputial separation in cohort 1B males.
Vaginal opening: There were no test item treatment-related effects on the mean age of vaginal opening in cohort 1B females. At 250 mg/kg/day and when compared with controls, there was an apparent delay in mean age at vaginal opening. The difference was mainly related to one female (L24695) which was positive for this test on Day 78 only (remnants of vaginal membrane still present).

Refer to the attached document (DOC 7) in the field below 'Attached background material’.

Cohort 2A:
Balanopreputial separation: There were no effects on the mean age of balanopreputial separation in cohort 2A males.
Vaginal opening: There were no effects on the mean age of vaginal opening in cohort 2A females.

Refer to the attached document (DOC 11) in the field below 'Attached background material’.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Pups during lactation (Day 4 p.p.): In test item-treated groups and when compared with controls, there were slightly lower AGD/BW1/3 ratio in male pups from 100 mg/kg/day onwards, reaching statistical significance at 250 mg/kg/day only (2.83 vs. 2.95 in controls, p<0.01). In the absence of effects on mean age at balanopreputial separation (cohorts 1A, 1B and 2A) and sexual maturation, this finding was considered to be of non-toxicological significance.
There were no effects in female pups.
Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Pups: pups at Day 13 p.p: There were no areolae or nipples in day 13 p.p. male pups.
Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):

Pups:
Non selected pups:
There were not test item-related organ weight differences.
The few organ weight changes were not considered to be test item related because they were of insufficient magnitude, were not dose-related and/or had no macroscopic correlates.
Refer to the attached document (DOC21) in the field below 'Attached background material’.

Cohort 1A:
Decreased absolute and relative-to-body thymus weights were recorded in males treated at 250 mg/kg/day (-19% in absolute weight and -14% in relative-to-body weight; p<0.01 or 0.05). In the absence of gross or microscopic correlates and since it was not seen in females, these differences were considered to be non-adverse and of low toxicological importance. There may be a contribution of stress in this difference.
The other organ weight changes were not considered to be test item-related because they were of insufficient magnitude, were not dose-related and/or did not correlate to microscopic findings. This included the higher relative-to-body liver weights in males treated at 100 or 250 mg/kg/day.

There were lower absolute and relative-to-body mesenteric lymph node weights in males treated at 100 mg/kg/day and lower absolute mesenteric lymph node weights in males treated at 250 mg/kg/day (p<0.01 or 0.005). There was also a trend toward a decrease in mandibular lymph node weights at 250 mg/kg/day in males and females (p<0.05). The inter-individual variability was high and there were no clear microscopic correlates. Consequently, and in view of their low magnitude, these changes were considered to be probably unrelated to the test item administration.

Refer to the attached documents (DOC 4 and DOC 5) in the field below 'Attached background material’.

Cohort 1B:
There were no test item-related organ weight changes.
The few organ weight differences were not considered to be test item-related because they were of insufficient magnitude and/or were not dose-related.
Refer to the attached document (DOC 7 and DOC 8) in the field below 'Attached background material’.

Cohort 2A:
There were no test item-related organ weight changes. The few organ weight differences were not considered to be test item related because they were considered to be related to the lower body weights in high-dose males, of insufficient magnitude and/or were not dose-related.
Refer to the attached document (DOC 11 and DOC 13) in the field below 'Attached background material’.

Cohort 2B:
There were no test item-related organ weight changes.
The few organ weight changes were not considered to be test item related because they were considered to be related to the decrease in body weight, were of insufficient magnitude, were not dose-related and/or had no macroscopic/microscopic correlates.
Refer to the attached document (DOC 16 and DOC 17) in the field below 'Attached background material’.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pups:
Macroscopic post-mortem observation in F1 pups: In test item-treated groups and when compared with controls, there were no evidence of any test item treatment-related findings in pups culled on Day 4 p.p. or sacrifice after weaning.
At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a secondary consequence of lack of maternal care, as previously mentioned.
Refer to the attached document (DOC 3) in the field below 'Attached background material’.

Non selected pups:
There were no gross changes.
Refer to the attached document (DOC 22) in the field below 'Attached background material’.

F1 cohorts:
Cohort 1A:
The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age. This included the small testes and epididymides seen in occasional males treated at 100 mg/kg/day or at 250 mg/kg/day.
Refer to the attached documents (DOC 4 and DOC 6) in the field below 'Attached background material’.

Cohort 1B:
There were no test item-related gross changes. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
Refer to the attached documents (DOC 7 and 9) in the field below 'Attached background material’.

Cohort 2A:
A black discoloration was noted in the forestomach from 1/10 high-dose females that correlated with slight ulcer. This was related to test item administration although seen at low incidence and severity, in view of the results in the other cohorts. The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
Refer to the attached document (DOC 11) in the field below 'Attached background material’.

Cohort 2B:
There were no test item-related gross changes. The few isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.
Refer to the attached documents (DOC 16 and 18) in the field below 'Attached background material’.

Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 cohorts:
Cohort 1A:
Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate (seen with higher severity than controls in test item-treated males) and edema (females only) were noted in forestomach from some males and/or females treated at 250 mg/kg/day. In one male treated at 250 mg/kg/day, minimal focal myofiber degeneration was recorded. In one other male and in one female treated at 250 mg/kg/day, minimal to slight focal ulcer was also noted. In addition, minimal to slight hyperplasia of squamous cells and hyperkeratosis were noted in a few females treated at 40 or 100 mg/kg/day, accompanied by minimal ulcer, mixed infiltrate and edema in one female (L24617) treated at 100 mg/kg/day.
The spectrum of lesions including squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or myofiber degeneration, and ulcer in one male and in one female, was considered to be adverse at 250 mg/kg/day in view of the severities (up to moderate) and of the nature of these lesions (subacute/chronic inflammation).
The ulcer seen in female L24617 treated at 100 mg/kg/day was accompanied by mixed infiltrate and edema. These findings were also considered to be adverse.
In view of the very low severity of hyperkeratosis and hyperplasia in females treated at 40 mg/kg/day which were of similar magnitude as those noted in control males and of the absence of associated ulceration, these findings were considered to be non-adverse at 40 mg/kg/day. No test item-related changes were noted in males treated at 40 mg/kg/day.

No changes were noted in the bone marrow.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls. This included, among others, minimal to severe atrophy/degeneration of tubules in testes from 1/20 males treated at 100 mg/kg/day and in 4/20 males treated at 250 mg/kg/day. These changes were unilateral in all affected males which suggested a spontaneous origin.
There were no test item-related changes in the male or female reproductive organs nor in nervous system..
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle except in 2 control females (L24514 and L24518) that had low cellularity and presence of intermediate cells which were inconsistent with the microscopic cycle (estrus and metestrus). These discrepancies were considered not to compromise the validity of these data since they occurred in controls and were seen at low incidence (2/20).

Refer to the attached document (DOC 4) in the field below 'Attached background material’.

Cohort 1B:
There were no test item-related changes.
The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changesfound in controls.
Refer to the attached document (DOC 7 and DOC 10) in the field below 'Attached background material’.

Cohort 2A:
Slight focal ulcer was noted in the forestomach of one high-dose female, together with edema, mixed inflammation, hyperkeratosis, acanthosis and hemorrhage, similarly to the changes noted in parents and cohort 1A. This finding was thus considered to be related to the test item administration and was considered to be adverse in spite of the isolated incidence, focal distribution and low severity of this change.
The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
Refer to the attached document (DOC 11 and DOC 14) in the field below 'Attached background material’.

Neurohistopathology:
Cohort 2A:
Brain weights: There were no test item-related brain weight differences. The few observed differences were minimal and not dose-related.
Semi-quantitative microscopic examination: The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulb, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas. There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.
Morphometric measurements of regions of interest in cerebral cortex (level 3), hippocampus (level 4) and cerebellum (level 7): A minimum of six (up to ten) brains/group/sex considered as appropriate were obtained for this histomorphometric evaluation.
There were no differences between controls and high-dose groups for the measurements performed.

The sensory cortex thickness (presented as L3 L2+6 in the table) was investigated in low- and intermediate-dose males because of lower results in test item-treated males at 250 mg/kg/day.
This measurement was statistically lower only in mid-dose males when compared with controls (-7%; p<0.05). The thickness in control males was 3755.65 µm (SD : 223 µm) while that of high-dose males was 3612.58 µm (SD: 85 µm) and 3501.02 µm in mid-dose males (SD: 118 µm). There was no dose relationship and the differences were considered to be of low magnitude. The relationship to test item of these differences was thus considered to be unrelated to the test item. No statistically significant changes were recorded in females
Refer to the attached document (DOC 11, DOC 13, DOC 14 and DOC 15) in the field below 'Attached background material’.

Cohort 2B:
There were no test item-related microscopic findings. The few microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings.
Refer to the attached document (DOC 16 and DOC 19) in the field below 'Attached background material’.

Neurohistopathology
Brain weights: There were no test item-related brain weight differences. The few observed differences were minimal and not dose-related.

Semi-quantitative microscopic examination: The examination by the pathologist of the HE-stained multiple brain sections allowed examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum. There were no test item-related findings in these areas. There were no effects in the eyes (retina and optic nerve), peripheral nerve, muscle or spinal cord.

Morphometric measurements of regions of interest in cerebral cortex (level 3), hippocampus (level 4) and cerebellum (level 7): A minimum of 6 (up to 10) brains/group/sex considered as appropriate were obtained for this histomorphometric evaluation (see text-table below).There were no differences between controls and high-dose groups for the measurements performed.
The motor cortex thickness (presented as L3 L1+5 in the table) was investigated in low- and intermediate dose females because of lower results in test item-treated females. This measurement was statistically lower only in mid-dose females when compared with controls (-8%; p<0.05).
The thickness in control females was 4035.61 µm (SD : 212 µm) while that of high-dose females was 3775.40 µm (SD : 289 µm) and 3722.38 µm in mid-dose females (SD : 144 µm). There was no dose-relationship. The relationship to test item of this difference was thus considered to be unrelated to the test item. No statistically significant changes were noted in males.
Refer to the attached document (DOC 16, DOC 17, DOC 19, DOC 20) in the field below 'Attached background material’.

Other effects:
no effects observed
Description (incidence and severity):
Reproductive fonction:
Cohort 1A: In test item-treated groups and when compared with controls, there were no effects on sperm analysis data in cohort 1A males.
Cohort 1A: There were no effects on mean estrous cycle parameters in cohort 1A females.
Refer to the attached document (DOC 4) in the field below 'Attached background material’.

Thyroid hormones (T4 and TSH)

Non-selected F1 offspring Day 4 p.p. and Day 22 p.p.:
There were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentrations in non-selected F1 pups (Day 4 and 22 p.p.).
Refer to the attached document (DOC 3) in the field below 'Attached background material’.

F1 cohorts:
Cohort 1A:
There were no effects on mean plasma thyroid hormones (T4 and TSH) concentrations in cohorts 1A animals (males or females).
Refer to the attached document (DOC 4) in the field below 'Attached background material’.


Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohorts 2A:
Auditory startle test: There were no effects on mean latency times and amplitudes of the responses measured during the auditory startle reflex test.

Reactivity to manipulation or to different stimuli: Cohort 2A animals were tested once or twice between Days 65 and 72 p.p. There was no abnormal reactivity to manipulation or to different stimuli.

Detailed clinical examination:
"touch escape" or "ease of removal from the cage" (reactivity to handing) were normal,
fur appearances were normal when compared with controls,
there were no excessive grooming, defecation, urination in treated groups when compared with the control group,
there were no salivation, no lacrimation, no piloerection and no palpebral closure,
all pups had a normal pupil size (myosis) at examination and there were no exophthalmos,
all pups had a normal gait, posture, behaviour and breathing,
there were no tremors, no twitches, no clonic/tonic convulsions, no ataxia, no hypoactivity, no hyperactivity, no hypotonia (except one female at 40 mg/kg/day) and no stereotypies (except one control female and one female at 250 mg/kg/day).

Reactivity to stimuli
visual stimulus response and pupillary reflex were normal in all groups,
auditory startle reflex was normal in all groups,
forelimb grip strength was normal in all groups.

Motor activity: In test item-treated groups and when compared with controls, there were no effects on mean number of horizontal movements and rearings.

Refer to the attached documents (DOC 11 and DOC 12) in the field below 'Attached background material’.
Developmental immunotoxicity:
not examined
Similar to the parent generation, the main toxic effects observed on the F1 cohorts are effects on CNS such as hypoactivity and half closed eyes and stomach inflammation.
Furthermore no developmental neurotoxicity effects were observed in the cohorts 2A and 2B.


Other results not detailed on the different sections above:
- Parental generation offspring:
Sex ratio (Day 21 p.p).
In test item-treated groups and when compared with controls, there were no effects on sex-ratio (percentage of male pups).
Refer to the attached document (DOC 3) in the field below 'Attached background material’.

- Cohorts F1 results:
Primordial follicles:
Cohort 1A: There was a minimal difference between the high-dose and the control groups, with a total number of 7.24 and 8.94 primordial follicles per section on PCNA-stained slides respectively. However, it has to be compared to the total number of primordial follicles in control parental cohort (5.06). This minimal decrease in high-dose females versus controls was of low magnitude (-19%) and displayed a high inter-individual variability, as suggested by the high standard deviations. Thus a relationship to test item administration was considered to be unlikely.
Refer to the attached document (DOC 4) in the field 'field below 'Attached background material’.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Cohort 1A: clinical signs (CNS) in both sexes from 100 mg/kg/day (hypoactivity at 100 mg/kg/day and half-closed eyes in females at 250 mg/kg/day), decrease of body weight in both sexes at 250 mg/kg/day and forestomach inflammation from 100 mg/kg/day in females and at 250 mg/kg/day in both sexes. Cohort 1B: clinical signs (CNS) in both sexes from 100 mg/kg/day (hypoactivity). Cohort 2A: clinical signs (CNS) in both sexes from 100 mg/kg/day (hypoactivity) and forestomach inflammation at 250 mg/kg/day.
Reproductive effects observed:
no

Additional table of results:

Tables of results for P generation

Parental general effects:

Clinical signs:

Clinical signs in terminated as scheduled P generation males

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

23

23

24

23

Ptyalism

 

4

(Days 53-90)

24

(Days 10-131)

23

(Days 1‑131)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

24

(Days 62-130)

23

(Days 1-131)

Half-closed eyes (episodes, 1 to 2 hours after dosing)

 

 

 

3

(Days 70-120)

Staggering gate (1 to 2 hours after dosing)

 

 

 

1

(Days 105‑109)

(): in brackets, period of occurrence.

Clinical signs in terminated as scheduled P generation females

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

22

24

22

22

Ptyalism

1

(Day 111)

2

(Days 16-75)

22

(Days 6-121)

22

(Days 1-126)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

19

(Days 72-119)

22

(Days 1-121)

Half-closed eyes (episodes, 1 to 2 hours after dosing)

 

 

 

4

(Days 70-73)

Staggering gate/tonic seizures (1 to 2 hours after dosing)

 

 

 

1

(Day 99)

Histopathology:

Main microscopic lesions (incidences) - P generation

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

40

100

250

0

40

100

250

Number of animals per group

23

23

24

23

22

24

22

22

Forestomach; ulcer

 

 

. grade 2

-

-

-

-

-

-

-

1

Forestomach; hyperplasia; squamous cell

 

 

. grade 1

-

-

1

3

-

-

2

6

. grade 2

-

-

-

10

-

-

-

3

. grade 3

-

-

-

2

-

-

-

1

Forestomach; hyperkeratosis

 

 

. grade 1

-

-

2

7

-

-

3

6

. grade 2

-

-

-

11

-

-

-

4

. grade 3

-

-

-

1

-

-

-

-

Forestomach; hemorrhage

 

 

. grade 1

-

-

-

2

-

-

-

-

Forestomach, infiltrate; mixed cell

 

 

. grade 1

-

-

-

3

-

-

-

-

. grade 2

-

-

-

5

-

-

-

2

Forestomach; edema

 

 

. grade 1

-

-

-

4

-

-

-

-

. grade 2

-

-

-

4

-

-

-

-

. grade 3

-

-

-

-

-

-

-

1

Forestomach; fibrosis

 

 

. grade 1

-

-

-

1

-

-

-

-

. grade 2

-

-

-

2

-

-

-

-

. grade 3

-

-

-

-

-

-

-

1

Bone marrow; increased cellularity

 

 

. grade 1

1

-

1

7

-

-

-

1

. grade 2

-

-

-

1

-

-

-

1

Bone marrow; increased myeloid/erythroid ratio

 

 

. grade 1

-

-

-

-

-

-

-

1

. grade 2

-

-

-

-

-

-

-

1

-: not observed.

 

Mating and fertility data:

Mating and fertility dataof the P generation

 

Dose level (mg/kg/day)

0

40

100

250

HCD

Number of animals paired (M + F)

23+23

24+24

23+23

24+24

171 F

Number of females mated

23

24

22

24

171

Female mating index (%)

100

100

95.7

100

[100]

Mean number of days taken to mate (days ± SD)

3.1 ± 2.0

3.2 ± 2.3

2.6 ± 1.2

2.9 ± 1.9

/

Number of pregnant females

23

23

19

23

165

Female fertility index (%)

100

95.8

86.4

95.8

91.7 ‑ 100]

M: males; F: females; HCD: Historical Control Data (two-generation reproduction toxicity studies in rats, OECD 416 guideline, October 2009 to February 2016, n = 7 studies), [min. - max.], /: not reported.

No statistically significant differences from controls.

 

Reproductive data:

 

Reproductive data of the P generation

 

Dose level (mg/kg/day)

0

40

100

250

HCD

Number of pregnant females

23

23

19

23

165

Number of females with liveborn

22

23

19

22

164

Gestation index (%)

95.7

100

100

95.7

[95.8 - 100.0] (a)

Mean duration of gestation

(days ± SD)

22.1 ± 0.4

22.0 ± 0.2

22.2 ± 0.5

22.1 ± 0.4

[21.0 - 22.0] (a)

Mean percentage of pre‑implantation loss

(% ± SD)

8.9 ± 11.4

5.7 ± 10.0

5.1 ± 7.9

10.4 ± 16.3

[5.5 – 22.5] (b)

Mean percentage of post‑implantation loss

(% ± SD)

15.4 ± 10.1

22.0 ± 18.1

26.4 ± 19.1

24.1 ± 25.3

/ (a)

Live birth index

(% ± SD)

98.0 ± 5.6

96.7 ± 8.0

90.9 ± 20.6

95.6 ± 11.2

100 (a, c)

Sex ratio on Day 1p.p.

(% ± SD)

43.8 ± 12.8

42.8 ± 15.8

46.8 ± 18.2

50.1 ± 22.7

[50.3 - 52.5] (a)

(a): HCD: Historical Control Data (two-generation reproduction toxicity studies in rats, OECD 416 guideline, October 2009 to February 2016, n = 7 studies); /: not reported; [min. - max.]. (a): not fully representative (different calculation in HCD).

(b): HCD: Historical Control Data (embryo-fetal development in rats, ICH and OECD 414 guidelines, August 2016 to December 2017, n = 11 studies); [min. - max.]. (a): not fully representative (different calculation in HCD).

(c): not fully representative (different calculation in HCD)

Sex ratio: percentage of male pups.

No statistically significant differences from controls.

Sperm analysis:

Sperm analysis data in males from the P generation

 

Dose level (mg/kg/day)

0

40

100

250

% of motile epididymal sperm ± SD

98.5 ± 3.5

97.5 ± 4.8

97.3 ± 5.9

95.1 ± 7.2

% of normal sperm ± SD

95.2 ± 1.5

/

/

95.3 ± 1.9

Mean number of epididymal sperm (106/cauda of epididymis) ± SD

133 ± 31

/

/

137 ± 29

Mean number of epididymal sperm (106/g of cauda of epididymis) ± SD

373.3 ± 64.7

/

/

414.9 ± 93.5

Mean number of testicular sperm heads (106/g testis) ± SD

115.3 ± 16.7

/

/

120.1 ± 19.6

Daily sperm production rate
(106/g testis/day) ± SD

18.9 ± 2.7

/

/

19.7 ± 3.2

/: not performed.

No statistically significant differences from controls.

 

Tables of results for F1 cohorts

Clinical signs:

Cohort 1A

Clinical signs in cohort 1A males (number of animal affected)

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

20

19

20

20

Ptyalism

 

 

20

(Days 1-73)

20

(Days 1-74)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

20

(Days 1-71)

20

(Days 1-74)

(): in brackets, period of occurrence.

Clinical signs in cohort 1A females (number of animal affected)

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

20

20

19

20

Ptyalism

 

1

(Days 64-65)

19

(Days 1-73)

20

(Days 1-74)

 

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

19

(Days 1-69)

20

(Days 1-72)

Half-closed eyes (episodes, 1 to 2 hours after dosing)

 

 

 

20

(Days 56-61)

(): in brackets, period of occurrence.

Cohort 1B

Clinical signs in cohort 1B males (number of animal affected)

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

20

20

20

20

Ptyalism

 

 

20

(Days 1-79)

20

(Days 1-79)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

19

(Days 1-79)

20

(Days 1-79)

(): in brackets, period of occurrence.

Clinical signs in cohort 1B females (number of animal affected)

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

20

20

20

20

Ptyalism

 

4

(Day 48-58)

20

(Days 1-79)

20

(Days 1-79)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

19

(Days 1-79)

20

(Days 1-78)

(): in brackets, period of occurrence.

 

Cohort 2A

Clinical signs in terminated as scheduled cohort 2A males

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

10

10

10

10

Piloerection

 

 

 

1

(Days 1-2)

Dyspnea

 

 

 

1

(Days 1-2)

Ptyalism

 

 

10

(Days 1-55)

10

(Days 1-56)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

 

7

(Days 1-51)

10

(Days 1-55)

Staggering gait (episodes, 1 to 2 hours)

 

 

 

1

(Day 1)

(): in brackets, period of occurrence.


 

Clinical signs in cohort 2A females (number of animal affected)

(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)

 

Dose level (mg/kg/day)

0

40

100

250

Number of animals terminated as scheduled

10

10

10

10

Loud breathing

 

 

 

1

(Day 11)

Ptyalism

 

 

10

(Days 12-56)

10

(Days 1-56)

Hypoactivity (episodes, 1 to 2 hours after dosing)

 

1

(Day 2)

6

(Days 1-55)

10

(Days 1-56)

(): in brackets, period of occurrence.

 

Histopathology:

Cohort 1A:

Main microscopic lesions (incidences) - cohort 1A

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

40

100

250

0

40

100

250

Number of animals per group

20

19

20

20

20

20

19

20

Forestomach; degeneration/necrosis; myofiber

 

 

. grade 1

-

-

-

1

-

-

-

-

Forestomach; ulcer

 

 

. grade 1

-

-

-

1

-

-

1

-

. grade 2

-

-

-

-

-

-

-

1

Forestomach; hyperplasia; squamous cell

 

 

. grade 1

-

-

-

5

-

1

-

6

. grade 2

-

-

-

2

-

-

1

1

. grade 3

-

-

-

-

-

-

-

1

Forestomach; hyperkeratosis

 

 

. grade 1

-

-

-

6

-

2

2

9

. grade 2

-

-

-

3

-

-

-

2

Forestomach; hemorrhage

 

 

. grade 1

-

-

-

1

-

-

-

-

Forestomach, infiltrate; mixed cell

 

 

. grade 1

2

-

-

1

-

-

1

1

. grade 2

-

-

-

1

-

-

-

1

Forestomach; edema

 

 

. grade 1

2

-

-

1

-

-

1

1

. grade 2

-

-

-

-

-

-

-

2

-: not observed.


 

Cohort 2A:

Main gross changes (incidences) – cohort 2A

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose-level (mg/kg/day)

0

40

100

250

0

40

100

250

Number of animals per group

10

10

10

10

10

10

10

10

Forestomach; black discoloration

-

-

-

-

-

-

-

1

-: not observed

 

 More complete data and tables are attached in the field below 'Attached background material'.


Conclusions:
Based on this study the main effects observed in parents and in F1 cohorts from 100 mg/kg/day are clinical signs such as hypoactivity and half-closed eyes showing effects on central nervous system (CNS). Inflammation of the forestomach was also noted in parents and in F1 cohorts (cohorts 1A and 2A). This inflammation was considered as adverse at 250 mg/kg/day in parents and in cohort 2A and from 100 mg/kg/day in cohort 1A for females. The data on parents did not show effect on reproduction/fertility. For P generation offspring, there was an apparent, but not statistically significant, dose-related decrease of mean litter size at the two higher doses and a statistically significant increase, but not dose-related, in the number of dead, missing and/or cannibalized pups at the same doses. At necropsy of pups found dead there were higher numbers of pups with absent of milk in the stomach. This finding was considered to be a consequence of lack of maternal care. When taken together, these finding (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day. There is an absence of direct developmental effect on pups. Furthermore, no malformations were observed in pups and there is an absence of test item treatment-related adverse findings in cohorts 2A and 2B regarding developmental neurotoxicity.

Executive summary:

In this OECD 443 study, sexually-mature male and female Sprague-Dawley rats (parental (P) generation) (four groups of 24 males and 24 females) were exposed to graduated doses (0, 40, 100 or 250 mg/kg/day) of paramethoxyphenol starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for:

a) reproductive/developmental toxicity testing: cohort 1A and cohort 1B (without mating to produce a F2 generation),

b) developmental neurotoxicity testing: cohorts 2A and 2B.

 

The F1 offspring received further treatment with the test item (same dose levels than the P generation) from weaning to adulthood or sacrifice. The treatment schedules were the following: daily from weaning (Day 22 p.p.) until euthanasia (from Days 92 to 101 p.p.) for cohorts 1A and 1B and daily from weaning (Day 22 p.p.) until euthanasia (on Days 75 to 78 p.p.) for cohort 2A.

 

Examination:

 

Parents and F1 generation: For parental and F1 generations, clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. P generation males and females were paired until mating was obtained or 14 days had elapsed. Females from the P generation were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the F1 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. When feasible, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter (no culling for litter with less than 10 pups). Pup physical development was assessed at designated end-points.

 

Cohorts: Cohorts 1A and 1B: Animals were selected for assessment of effects upon reproductive systems and of general toxicity. Estrous cycle stages were determined daily for all cohort 1A females, after the onset of vaginal patency, until the first cornified smear was recorded (estrus) and for at least two weeks before the end of the treatment.

Cohort 2A: On Day 22 p.p., 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least one pup; randomly selected) were selected for neurobehavioral testing.

 

Terminal examinations: A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and not selected F1 pups on Day 22 p.p.). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The ratio of organ weight to body weight was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and a complete list of tissues from animals of all or control-/high-dose groups.

 

P generation and cohort 1A: The first 10/sex/group surviving animals were deprived of food for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cells evaluation was performed on surviving males from all or control-/high-dose groups: motility, morphology, sperm cell heads count in testicular/epididymal tissues.

 

Cohorts 2A and 2B: neurohistopathology was performed for all high-dose and control animals of each sex following completion of neurobehavioral testing (between Days 75 and 78 p.p. for cohort 2A animals and on Day 22 p.p. for cohort 2B animals).

 

Thyroid hormones levels (P generation, F1 culled pups and cohort 1A animals): prior to blood sampling the animals were not deprived of food. Blood samples were taken on Day 4 p.p. (F1 culled pups) for measurements of thyroid hormone (T4) levels and, on Day 22 p.p. (F1 pups not selected for cohorts) and at termination from the first ten surviving males/group and the first ten surviving females/group (P and cohort 1A animals), for measurements of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.

 

Results 

P generation 

 

Mortality: there were no test item treatment-related deaths in male or female animals from the P Generation.

 

Clinical signs (males and females):  at 250 mg/kg/day, half-closed eyes recorded in a few animals was considered to be test item treatment-related. From 100 mg/kg/day, hypoactivity recorded in almost all treated rats was considered to be test item treatment-related. From 40 mg/kg/day, ptyalism (a common finding usually observed after repeated gavage administrations) was observed at increased dose level incidences and was considered to be test item treatment-related but non adverse.

 

Mean body weight: males: at 250 mg/kg/day, there was a low mean body weight which was considered to be test item treatment-related and adverse. At 100 and 40 mg/kg/day, there were no effects.

Females: there were no effects during the premating, gestation or lactation periods.

 

Mean body weight change: males: at 250 mg/kg/day, there was a low mean body weight gain which was considered to be test item treatment-related and adverse. At 100 and 40 mg/kg/day, there were no relevant findings.

Females: at 250, 100 and 40 mg/kg/day, there were no relevant findings during the premating, or gestation. In all test item-treated groups, lower mean body weight gains during the lactation period (Days 1 to 4 p.p.) were recorded. These findings were considered to be test item treatment-related but not adverse based on returns towards control values from Days 4 to 7 p.p.

 

Food consumption: males: at 250, 100 and 40 mg/kg/day, there were no effects. Females: during the premating period and at 250 mg/kg/day, there was a low mean food consumption. This finding was considered to be test item treatment-related and adverse but of moderate toxicological importance. During the gestation period, there were no significant effects. During the lactation period and at 250 mg/kg/day, there was a low mean food consumption. This finding was considered to be test item treatment-related and adverse but of moderate toxicological importance. At 100 and 40 mg/kg/day, there were no effects.

 

Estrous cycle: at 250 mg/kg/day, there was a high mean number of days of diestrus and a low mean number of days of proestrus. This finding was considered to be test item treatment-related but not adverse as all values remained within the range of Historical Control Data. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Mating and fertility: there were no test item dose-related effects on mating (mating index), mating behavior (time taken to mate) and fertility (fertility index).

 

Delivery data: there were no test item treatment-related significant effects on mean duration of gestation, number of females with live born, percentage of pre- and post-implantation loss, pup live birth index, and pup sex ratio.

 

Laboratory investigations: there were no relevant effects on hematology and coagulation parameters and on blood biochemistry. There were no test item treatment-related findings on urinalysis parameters. There were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentration. There were no test item treatment-related effects on sperm analysis.

 

 

P generation offspring (pre-weaning F1 pups):

 

Mortality: at 250 and 100 mg/kg/day and when compared with controls, there was a dose-related decrease in mean litter size with values below the lower limit of the Historical Control Data and a statistically significant increase in the number of dead, missing and/or cannibalized pups on Days 1-4 p.p. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data. At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach. This finding was considered to be a consequence of lack of maternal care. At 40 mg/kg/day, there were no significant effects. When taken together, these findings observed at the two higher doses (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity.  Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day. There were no direct effects of the test-item on pup development.

 

Clinical signs: in test item-treated groups and when compared with controls, there were no test item treatment-related significant effects.

 

Body weight: there were no toxicologically significant effects.

 

Anogenital distance (Day 4 p.p.): there was a dose-related decrease in AGD/BW1/3ratio in male pups from 100 mg/kg/day onwards, reaching statistical significance at 250 mg/kg/day. In the absence of effects on mean age at balanopreputial separation (cohorts 1A, 1B and 2A), this finding was considered to be of non-toxicological significance.

 

Areolae and nipples (Day 13 p.p.): there were no areolae or nipples in Day 13 p.p. male pups.

Sex ratio (Day 21 p.p): there were no statistically significant differences.

 

Thyroid hormones: there were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentrations in no-selected pups (Day 4 or 22 p.p.)

 

Macroscopic post-mortem observation: there were no evidence of any test item treatment-related findings in found dead, pups culled on Day 4 p.p. or pups sacrifice after weaning. 

 

Cohort 1A

 

Mortality: there were no test item treatment-related unscheduled deaths.

 

Clinical signs: males: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were observed in all animals. These findings were considered to be test item treatment-related. Females: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were observed in all animals. At 250 mg/kg/day half-closed eyes were noted in all animals. These findings were considered to be test item treatment-related.

 

Mean body weight: males: at 250 mg/kg/day, there were episodes of lower mean body weight after weaning which were considered to be test item treatment-related and‑adverse. At 100 and 40 mg/kg/day, when compared with control, there were no effects. Females: at 250 mg/kg/day, there was a lower mean body weight after weaning which was considered to be test item treatment-related and-adverse. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Mean body weight change: at 250 mg/kg/day, in both sexes and when compared with controls, there were lower mean body weight gains after weaning. This finding was considered to be test item treatment‑related but non-adverse. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Food consumption: there were no significant effects on male or female mean food consumptions.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 1A animals and no effects on the mean time from vaginal opening to first oestrous in cohort 1A females.

 

Estrous cyles: there were no effects on mean estrous cycle parameters in cohort 1A females.

 

Laboratory investigations: there were no relevant effects on hematology and coagulation parameters and on blood biochemistry. There were no test item treatment-related findings on urinalysis parameters. There were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentration. There were no test item treatment-related effects on sperm analysis.

 

 

Cohort 1B

 

Mortality: there were no unscheduled deaths.

 

Clinical signs: males and females: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were recorded. These findings were considered to be test item treatment-related. At 40 mg/kg/day, there were no test item treatment-related findings.

 

Body weight and body weight change: males: there were no effects on mean body weights or mean body weight changes. Females: there were no relevant effects on mean body weights or mean body weight changes.

 

Food consumption: there were no relevant test item treatment-related effects on mean food consumption.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 1B animals.

 

Cohorts 2A

 

Mortality: there were no unscheduled deaths in cohort 2A males or females.

 

Clinical signs: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were recorded. These findings were considered to be test item treatment-related. At 40 mg/kg/day, there were no test item treatment-related findings.

 

Body weight: males: there were no effects on mean body weight. Females: at 250 mg/kg/day, there was a lower mean body weight after weaning. This finding was considered to be test item treatment-related but of minimal toxicological importance [based on the return towards control values from Day 22 (Day 43 p.p.)]. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Body weight change: at 250 mg/kg/day, in both sexes, there were lower mean body weight gains after weaning. This finding was considered to be test item treatment-related but of minimal toxicological importance [based on the return towards control values from Days 8-15 (Days 29-36p.p.)]. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Food consumption: there were no effects on mean food consumption in cohort 2A animals.

 

Neurobehavioral testing:

.  Auditory startle test: there were no effects.

.  Functional Observation Battery (FOB): there was no abnormal reactivity to manipulation or to different stimuli,

.  Motor activity: there were no effects on mean number of horizontal movements and rearings.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 2A animals

 

 

Pathology:

 

Parental generation: Decreased thymus weights were recorded in males treated at 250 mg/kg/day with no clear microscopic correlates. Test item-related white masses and/or thickening were noted in the forestomach from one female treated at 100 mg/kg/day and in two males and two females treated at 250 mg/kg/day. Dose-related adverse squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and fibrosis were noted in males and females treated at 250 mg/kg/day. In one female treated at 250 mg/kg/day, it was associated with slight ulcer. Non-adverse squamous cell hyperplasia and hyperkeratosis were noted in males and females treated at 100 mg/kg/day. Non adverse increased cellularity (with increased myeloid/erythroid ratio) was seen in the bone marrow from males and females treated at 250 mg/kg/day. This finding was considered to be of low toxicological importance. At quantitative evaluation of primordium follicles, there were no test item-related differences between the high-dose and the control groups.

 

Cohort 1A: Decreased thymus weights were recorded in males treated at 250 mg/kg/day. There were no test item-related gross changes. Dose-related adverse squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate and edema were noted in the forestomach from males and/or females treated at 250 mg/kg/day, and in females treated at 100 mg/kg/day. In one male treated at 250 mg/kg/day, minimal focal myofiber degeneration was recorded. In one other male, in one female treated at 250 mg/kg/day and in one female treated at 100 mg/kg/day, minimal to slight ulcer was also noted. Non-adverse squamous cell hyperplasia and hyperkeratosis were noted in females treated at 40 mg/kg/day. No changes were noted in the bone marrow. At quantitative evaluation of primordium follicles, there was a minimal decrease in high-dose females when compared to controls. However, a relationship to test item administration was considered to be unlikely in view of the low magnitude of this difference that did not reach statistical significance and of the high inter-individual variability. 

 

Cohort 1B: There were no test item-related organ weight or gross changes. There were no test item-related microscopic findings.

 

Cohort 2A: There were no test item-related organ weight changes. A black discoloration was noted in the forestomach from 1/10 high-dose females that correlated with slight ulcer together with edema, mixed inflammation, hyperkeratosis, acanthosis and hemorrhage. This was related to test item administration although seen at low incidence and severity, in view of the results in the other cohorts and was considered to be adverse. At neuropathology, there were no differences between controls and test item-treated groups for the measurements performed.

 

Cohort 2B: There were no test item-related organ weight or gross changes. There were no test item-related microscopic findings. At neuropathology, there were no differences between controls and test item-treated groups for the measurements performed.

 

Non selected pups: there was no reported mortality. There were not test item-related organ weight differences or gross changes. No microscopic examination was performed.

 

 

Conclusion

  

a) Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluded reproductive and developmental toxicity endpoints) was considered to be 40 mg/kg/day, based on clinical signs from 100 mg/kg/day, adverse histopathological changes in the forestomach from 100 mg/kg/day in females, decreased body weight at 250 mg/kg/day (both in males and females) and adverse histopathological changes in the forestomach at 250 mg/kg/day in males.

 

b) Reproductive/developmental toxicity testing:

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 250 mg/kg/day, based on the absence of direct test item treatment-related adverse findings in P generation, cohorts 1A or 1B animals. Decreased mean litter size, increase number of dead, missing and/or cannibalized pups were observed from 100 mg/kg/day and were considered as a secondary consequence of lack of maternal care in F1 offspring on Days 1-4 p.p.

 

c) Developmental neurotoxicity testing:

The No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 250 mg/kg/day based on absence of test item treatment-related adverse findings in cohorts 2A or 2B animals.

 

 

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The lowest NOAEL comes from the OECD 443 study with reliability 1. This study has been performed in 2019 under GLP compliance. No adverse reprotoxic effect related to the substance was observed at the highest tested dose.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Two studies are referenced under the section toxicity to the reproduction: an OECD 422 and an OECD 443 studies. Details on these studies are reported below.

OECD 422 study (Harlan, 2009):

This study was scored as Klimisch 1, GLP study without deviation, and it was chosen as supporting 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 HanRcc: WIST (SPF) 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, post-implantation 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). These effects were not dose dependent, within the range of historical control data and were considered as incidental findings. The NOAEL for reproduction/fertility toxicity was thus considered to be greater than 300 mg/kg/day.

Based on this study, Paramethoxyphenol is not considered to have an impact on reproduction or fertility.

 

OECD 443 study (CitoxLab, 2019b) :

This study was scored as Klimisch 1, GLP study without deviation, and it was chosen as key study. In this study, sexually-mature Sprague-Dawley rats (P generation) were exposed to graduated doses of paramethoxyphenol (0, 40, 100 and 250 mg/kg/day) starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for a) reproductive/developmental toxicity testing: cohort 1A and cohort 1B (without mating to produce a F2 generation) and for b) developmental neurotoxicity testing: cohorts 2A and 2B. The F1 offspring received further treatment with the test item from weaning to adulthood or sacrifice. For P generation, there were no test item treatment-related deaths in male or female animals. At 250 mg/kg/day, half-closed eyes were recorded in a few animals and from 100 mg/kg/day, hypoactivity was recorded in almost all treated rats. These findings were considered to be test item treatment-related and adverse since these effects were also noted in the cohorts: cohort 1A, 1B, and 2A. At 250 mg/kg/day, in males, there was a low mean body weight which was considered to be test item treatment-related and adverse. Dose-related adverse squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and fibrosis were noted in males and females treated at 250 mg/kg/day. Regarding reproduction parameters, there were no adverse effects on estrous cycle, no test item dose-related on mating, mating behavior and fertility and no test item treatment-related significant effects on mean duration of gestation, number of females with live born, percentage of pre- and post-implantation loss, pup live birth index, and pup sex ratio.

Based on the results of this study the NOAEL for systemic toxicity (excluded reproductive and developmental toxicity endpoints) was considered to be 40 mg/kg/day, based on clinical signs observed from 100 mg/kg/day in parents. The NOAEL for reproduction/fertility was considered to be 250 mg/kg/day based on an absence of adverse effect on estrous cycle and based on an absence of test item dose-related or statistically significant effect on mating index, mating behavior, fertility index, mean duration of gestation, number of females with live born, percentage of pre- and post-implantation loss, pup live at birth index, and pup sex ratio.

Short description of key information: Based on the OECD 422 test guideline study, the NOAEL for reproduction/fertility toxicity was considered to be greater than 300 mg/kg/day (the higher tested dose) based on the absence of effect on the reproduction parameters.

Based on the OECD 443 test guideline study, the NOAEL for reproduction/fertility toxicity was considered to be 250 mg/kg/day since there were no test substance-related adverse findings in P generation reproduction parameters.


Effects on developmental toxicity

Description of key information

Two prenatal developmental studies are available, an OECD 414 study performed in rats in 2013 and an OECD 414 study conducted in a second species (rabbits) in 2019. Furthermore an OECD 443 has been performed in rats in 2019.

 

Based on the OECD 414 study performed in 2013, specific malformations and variations were observed in rat fetuses at the highest tested dose in a context of a very marked (excessive) maternal toxicity (see also below the section 'Justification for classification or non classification'). 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 and, decreased mean body weight, mean body weight change and/or mean food consumption. At 400 mg/kg these findings were more severe, going beyond the 'High dose' as intended in OECD guideline 414 and thus in excess of the maximum tolerated dose. Therefore fetal observations at this dose level should not be considered for assessment. The findings noted here are only documentation purposes. In the 400 mg/kg/day group increased post-implantation losses (mainly as a consequence of increased early resorptions) occurred. Additionally, in such a context with excessive maternal toxicity, developmental delays (reduced affecting fetal body weight and ossification associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded.

In the second OECD 414 study conducted in rabbits in 2019, there were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss) whatever the administered doses up to 250 mg/kg/day. There were no adverse effects on mean fetal body weight, mean placental weight and sex ratio whatever the administered doses up to 250 mg/kg/day. There were no test item treatment-related variations or malformations at external and soft tissue examination of the fetuses whatever the administered doses up to 250 mg/kg/day. In all test item-treated group and when compared with controls, there were higher number of fetuses with non or incompletely ossified hyoid (cartilage present but not ossified). This finding was considered test item treatment-related, but not adverse (bone cartilage present). There were no test item treatment-related skeletal malformations whatever the administered doses.

The OECD 443 study performed in rats in 2019 gives additional data. Based on this study, there were no adverse effects on number of females with live born, percentage of pre- and post-implantation loss, pup live birth index, and pup sex ratio. At 250 mg/kg/day and 100 mg/kg/day and when compared with controls, there was a dose-related decrease in mean litter size with values below the lower limit of the Historical Control Data (11.7 and 10.4 vs. 11.8 , at 100 and 250 mg/kg/day respectively) and a statistically significant increase in the number of dead, missing and/or cannibalized pups on Days 1-4 p.p. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values of dead, missing and/or cannibalized pups, while they remained higher than Historical Control Data. At necropsy of pups found dead, there were higher numbers of pups with absence of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a consequence of lack of maternal care. These effects (decrease in mean litter size and increase in the number of dead, missing and/or cannibalized pups) are not considered to be a direct effect on development, but as an indirect effect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to CNS effects (hypoactivity and half-closed eyes) of the test material at 100 and 250 mg/kg/day. After culling on Day 4 p.p., there were no effects on mean litter sizes or lactation indexes. Furthermore, this study showed no statistically significant effects on pups clinical signs and body weight and at macroscopic post-mortem observation there was no evidence of any test item treatment-related findings in found dead pups, pups culled on Day 4 p.p or pups sacrifice after weaning. There was no effect on sperm analysis on P generation and in 1A cohort. There were no effects on sexual development and on estrous cycles on 1A,1B and 2A cohorts. Neurobehavioral tests did not reveal effects on 2A cohort. There were no test item-related in males and females reproductive organs nor in nervous system in P generation, 1A, 1B, 2A and 2B cohorts. Neurohistopathology did not reveal effects on brain in 2A and 2B cohorts. Based on the results of this study the NOAEL for developmental toxicity was considered to be 250 mg/kg/day, based on the absence of direct test item treatment-related adverse findings in P generation offspring and based on the absence of test item treatment-related developement findings in cohort 1A or 1B animals. Furthermore, developmental neurotoxicity testing revealed no test item treatment-related findings in cohorts 2A or 2B animals. Therefore, the NOAEL for developmental neurotoxicity was considered to be 250 mg/kg/day.

 

The developmental delays and the specific malformations observed in an OECD 414 study in rats at the highest tested dose (400 mg/kg/day) were not observed in the OECD 443 study performed in rats at the dose levels up to 250 mg/kg/day and in rabbits based on the OECD 414 conducted in 2019.

Link to relevant study records

Referenceopen allclose all

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
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed from 100 mg/kg/day with few animals affected. This effect was considered to be related to the treatment with the test item, but of minor toxicological significance. This effect was not considered as adverse. Hypoactivity and half-closed eyes were also observed from 100 mg/kg/day in some animals (14/24 for hypoactivity and 12/24 for half-closed eyes). These effect are transient. Plioerection, locomotory difficulties and sedation were observed from 200 mg/kg/day. Round back and tonic contraction were observed at 400 mg/kg/day. All these clinical signs were recorded with dose-related increased incidence/severity and were considered to be test treatment-related from 100 mg/kg/day and of toxicological significant and adverse from 200 mg/kg/day. These effects were considered to be very severe at the dose level of 400 mg/kg/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 and at the dose of 400 mg/kg/day, 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 toxicological significant at 400 mg/kg/day.

There were no effect on mean body weight or mean body weight change at 100 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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 (-63.0% 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.
Food efficiency:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
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 observed only at the dose level of 400 mg/kg/day and were considered to be toxicologically significant.
At the two other treated doses (100 and 200 mg/kg/day) and when compared with controls, there was no change in mean post-implantation loss.
When compared with controls, there was no change in mean pre-implantation loss whatever the treated doses.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
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 observed only at the dose level of 400 mg/kg/day and were considered to be toxicologically significant. There were two pregnant females with total resorptions in the group treated at 400 mg/kg/day.

At the two other treated doses (100 and 200 mg/kg/day) and when compared with controls, there was no increase in the mean number of resorptions.

Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant increase in mean number of early resorptions. There was no change in mean number of early resorptions at the two other tested doses (100 and 200 mg/kg/day). When compared with controls, there was no change in mean number of late resorptions whatever the tested doses.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg/day and when compared with controls, there was a statistically significant decrease in mean number of live fetuses per litter (11.1% vs. 13.8, p<0.05) corresponding mainly to early resorptions.
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
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. 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.
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 in some animals (transient effects), 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. No effect were observed at the two other tested doses (100 and 200 mg/kg/day).
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
There is a decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05) due to early resorptions in two females at 400 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (mean percentage of male fetuses).
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
There is a decreased mean number of fetuses per litter (11.1 vs. 13.8, p<0.05) due to early resorptions in two females at 400 mg/kg/day.
External malformations:
effects observed, treatment-related
Description (incidence and severity):

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 (Tera, Anasarca, Cleft lip, Cleft palate, Omphalocele, short trunk and short tail) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

The increase fetal incidence of external malformation was statistically significant for the following external malformations: Cleft lip, Cleft palate and Omphalocele.

Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
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 (Vertebra(e), multiple malformations ; Interparietal, unossified ; Skullcap, absent ; Palate, split ; Mandible, fused ; Cervical vertebra(e), fused arches ; Cervical vertebra(e), absent ; Thoracic vertebrae(e), absent ; Supernumerary lumbar vertebra ; Sacral vertebra(e), absent ; Caudal vertebra(e), absent ; Fused sternebrae ; Misshapen sternum ; Misshapen rib ; Absent rib ; Masshapen rib cage and Fused ribs) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data.

The increase in fetal incidence of each skeletal malformation was not statistically significant. However, the increase in the number of fetus affected at the higher tested dose (400 mg/kg/day) was statistically significant.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Soft tissues malformations:
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 (Cleft palate, Anophthalmia, and Misshapen inner ear) were considered to be related to the test item treatment when compared with the control group or to the Historical Control Data. The increase in fetal incidence was statistically significant for Anophthalmia.

Soft tissues variations:
When compared with the control group or Historical Control Data, there were no test item treatment-related soft tissues variations.

External variations:
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. The increase was not statistically significant.

Skeletal variations:
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 (Frontal, incomplete ossification ; Fontanel, enlarged ; Maxilla, incomplete ossification ; Premaxilla, incomplete ossification and Metacarpal(s), unossified) were considered to represent significant ossifications delay and to be related to the test item treatment. The increase in fetal incidence was statistically significant for the following skeletal variations: Frontal, incomplete ossification ; Fontanel, enlarged ; Maxilla, incomplete ossification ; Premaxilla, incomplete ossification and Metacarpal(s), unossified.
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. There was no effect at the two other tested doses 100 and 200 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Remarks:
Development
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
skeletal malformations
other: Soft tissues malformations, external and skeletal malformations.
Remarks on result:
other:
Remarks:
All the effects observed on fetuses: increase in number of live fetuses, decrease in mean fetal body weight, increases in variations (external and skeletal) and increase in malformations (external, soft tissues and skeletal) were observed only at the dose level of 400 mg/kg/day in a context of very marked (excessive) maternal toxicity.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: eye
external: face
external: tail
external: trunk
skeletal: skull
skeletal: sternum
skeletal: rib
skeletal: vertebra
visceral/soft tissue: gastrointestinal tract
Description (incidence and severity):
for more details see tables 3, 4, 5, 6, 7, 8 and 9.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

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 (piloerection, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption.

At 400 mg/kg/day 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. The developmental effects (variations and malformations) and the resorptions were only observed at the higher tested dose (400 mg/kg/day) in a context of very marked (excessive) maternal toxicity. These effects seem to be indirect effects as a secondary non-specific consequence of maternal toxicity effects (maternal toxicity too excessive at 400 mg/kg/day).

While the study was conducted according to the guideline and under GLP conditions, the high dose level was exceeded the guideline and therefore fetal endpoints at the 400 mg/kg/day dose level are not considered relevant for evaluation in this study (see more explanation in the endpoint summary 'Toxicity to reproduction').
Executive summary:

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 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.

Results

Clinical signs: Ptyalism was observed in all test item-treated groups and 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 significance from 200 mg/kg/day.

Mean body weight and mean body weight changes: At 200 mg/kg/day, there were a minimal decrease in mean body weight which resulted in a lower mean body weight gain. At 400 mg/kg/day, there was a marked mean body weight loss over the period of days 6 to 9 p.c., thereafter mean body weight gain returned toward control values and decreased again from days 15 to 20 p.c.. During all the treatment period, there were decreases in mean body weight. All these findings were considered to be test item treatment-related from 200 mg/kg/day and of toxicological significance at 400 mg/kg/day.

Mean food consumption: At 100 mg/kg/day, there was a minimal decrease in mean food consumption at initiation of the treatment period which returned to control values thereafter. At 200 mg/kg/day, there was a marked decrease in mean food consumption at initiation of the treatment period which returned to control values thereafter. At 400 mg/kg/day, there was a severe decrease in mean food consumption at initiation of the treatment period which never returned to control values. All these findings were considered to be test item treatment-related from 100 mg/kg/day and of toxicological significance from 200 mg/kg/day.

Necropsy and hysterectomy data: At necropsy, there were no test item treatment-related effects at macroscopic examination. At 400 mg/kg/day, there was a decrease in mean uterus weight and from 200 mg/kg/day a

decrease in mean carcass weight. These findings were considered to be treatment-related from 200 mg/kg/day and toxicologically significant at 400 mg/kg/day. At 400 mg/kg/day, there were increased mean implantation loss (corresponding mainly to early resorptions) and decreased mean number of fetuses per litter. All these findings were considered to be toxicologically significant.

Fetal examination: There were no effects on sex-ratio. At 400 mg/kg/day, there were marked decreases in mean fetal body weight and in mean number of live fetuses per litter. At fetal examination, there were increased litter and fetal incidences of variations (external and skeletal) and malformations (external, soft tissues and skeletal).

In conclusion, 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 (piloerection, locomotory difficulties and sedation) and, decreased mean body weight, mean body weight change and/or mean food consumption. At 400 mg/kg/day 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 of very severe (excessive) maternal toxicity, developmental delays (reduced affecting fetal body weight and ossifictaion associated) and malformations (mainly of the brain, skull, head and axial skeleton) were recorded. At 400 mg/kg/day the maternal toxicity was very severe and beyond the 'High dose' as intended in OECD guideline 414. Therefore fetal observations at this dose level should not be considered for assessment.

While the study was conducted according to the guideline and under GLP conditions, the high dose level exceeded the guideline and therefore fetal endpoints at the 400 mg/kg/day dose level are not considered relevant for evaluation in this study (see more explanation in the endpoint summary 'Toxicity to reproduction').

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 (piloerection, 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 upon no findings at this dose level. As noted earlier fetal observations at 400 mg/kg/day are not considered relevant for classification and labelling purposes in this study due to the excessive maternal toxicity in this dose level.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 November 2017 to 27 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Mequinol (paramethoxyphenol)
- EC N°: 205-769-8
- CAS N°: 150-76-5
- Description: White flakes
- Purity: higher than 99%.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Centre LAGO (Vonnas, France).
- From arrival at Citoxlab France, the animals were housed in a barriered rabbit unit.
- Receipt: upon their arrival at Citoxlab France, the animals were given a clinical examination to ensure that they were in good condition.
- Strain and sanitary status: New Zealand White, INRA, A 1077, Specific Pathogen Free (S.P.F.).
- Number: 96 time-mated female rabbits.
- Age at study initiation: at the beginning of the treatment period, the animals were approximately 18-20 weeks old. The females were sexually mature and primigravid.
- Weight at study initiation: at the beginning of the treatment period, the animals had a mean body weight of 3652 g (range: 3125 g to 4230 g).
- Fasting period before study: no
- Housing: The animals were individually housed in noryl cages (Tecniplast, 4200 cm², 65.3 x 65.3 x 45 cm). Individual housing was chosen to avoid endangering gestation, as rabbits of this age sometimes show antisocial behavior towards each other. The cages were suspended in batteries over trays with absorbant paper and were placed in numerical order on the racks. Each cage contained dumbbell and hay (batch No. 8537207) for the environmental enrichment of the animals.
- Diet (e.g. ad libitum): All animals had free access to breeding pelleted diet "type 110C", batch No. 17192 (SAFE, Augy, France).
- Water (e.g. ad libitum):The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: the animals were acclimated to the study conditions 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 groups: during the acclimation period the animals were allocated to the groups, according to a stratified procedure base on body weight recorded on Day 0 p.c. provided by the breeder, to ensure comparatively similar mean body weight of the groups.
- Identification: each animal was individually identified by an ear tattoo detailing its unique Citoxlab France identity number.
- Contaminant analyses: The batch of diet was analyzed by the Supplier for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet or drinking water at levels which could be expected to interfere with, or prejudice, the outcome of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C,
- Humidity (%): : 50 ± 20%,
- Air changes (per hr): : 8 h dark/16 h light,
- Photoperiod (hrs dark / hrs light): about 5 to 15 cycles/hour of filtered, non-recycled air.

The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously (recording devices equipped with alarm systems).
The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.


IN-LIFE DATES: From: 17 November 2017 to 29 December 2017.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
Name: Drinking water treated by reverse osmosis using ELIX 5.
The vehicle for preparation of test item dose formulations was selected in agreement with the Sponsor, based on previous experimental work and/or preliminary studies performed on the same substance
(Citoxlab France/Study Nos. 44827 TSL: Dose range-finding study by the oral route (gavage) in non-pregnant rabbits and 44828 RSL: Preliminary study of prenatal developmental toxicity by oral route
(gavage) in rabbits).
- Concentration in vehicle: Dose levels 0; 40; 100 and 250 which correspond to the following concentration in vehicle: 0; 4 mg/mL; 10 mg/mL and 25 mg/mL.
- Dosage-Volume: 10 mL/kg.

PREPARATION PROCEDURE OF FORMULATION:
Before preparing the dose formulations, the test item was ground, using a mortar and pestle. Then, the required quantities were weighed and 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, protected from light and delivered in brown flasks. Stability confirmed at concentrations ranging from 2 to 35 mg/mL.


The suitability of the preparation process was confirmed by analysis of the stability as described in Citoxlab France/Study No. 39418 AHS. Stability was also analyzed before instituting any significant changes in dose formulation preparation (e.g. change in the range of concentrations). In Citoxlab France/Study No. 39418 AHS, the stability of the dose formulations prepared in drinking water treated by reverse osmosis was confirmed for up to 11 days, at concentrations ranging from 2 to 35 mg/mL,
stored at room temperature and protected from light.

ADMINISTRATION:
The dose formulation was administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 10 mL/kg/day was used. This volume is the maximum recommended in pregnant rabbit for daily repeated administrations and for which Historical Control Data are available. The dose formulations were maintained under room temperature and protected from light throughout the dosing procedure. They were stirred for at least 15 minutes before administration and were maintained under continuous magnetic stirring throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle and validation of the analytical method: the High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of PARAMETHOXYPHENOL in dose formulation samples was validated at Citoxlab France. 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.

Determination of dose formulation stability: The suitability of the preparation process was confirmed by analysis of the stability as described in Citoxlab France/Study No. 39418 AHS. Stability was also analyzed before instituting any significant changes in dose formulation preparation (e.g. change in the range of concentrations).

Determination of PARAMETHOXYPHENOL concentration in dose formulations: The concentration of the test item in samples of each control and test item dose formulation prepared for use at the beginning and the end of the treatment period (two occasions) was determined. Whenever possible these analyses were performed prior to administration of the dose formulations to the animals.
Acceptance criterion: measured concentration = nominal concentration ± 10%.

Details on mating procedure:
Mating: the females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.

Mating of females (Day 0 p.c)
First females: 15 november 2017
Last female: 30 november 2017
Duration of treatment / exposure:
Daily exposure Day 6 to Day 28 p.c.
Frequency of treatment:
Daily.
Duration of test:
Day 6 to Day 28 p.c., inclusive.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
The test item concentrations in the administered dose formulations analyzed at the beginning and the end of the treatment remained within an acceptable range of variations (-3.6% to +1.0%) when compared to the nominal values (± 10% of the nominal concentrations).
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
The test item concentrations in the administered dose formulations analyzed at the beginning and the end of the treatment remained within an acceptable range of variations (-3.6% to +1.0%) when compared to the nominal values (± 10% of the nominal concentrations).
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
The test item concentrations in the administered dose formulations analyzed at the beginning and the end of the treatment remained within an acceptable range of variations (-3.6% to +1.0%) when compared to the nominal values (± 10% of the nominal concentrations).
No. of animals per sex per dose:
24 mated female New Zealand White rabbits per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
The objective of this study was to evaluate the potential toxic effects of the test item, MEQUINOL (PARAMETHOXYPHENOL), on the pregnant female rabbit and, on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits [from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum (p.c.) inclusive)].

Three groups of 24 mated female New Zealand White rabbits received the test item, Mequinol (paramethoxyphenol), daily from gestation Day 6 to Day 28 p.c., by gavage, at dose levels of 40, 100 or 250 mg/kg/day. Another group of 24 mated females received the vehicle (drinking water treated by reverse osmosis), under the same experimental conditions and acted as a control group.
The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., females were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, uterine scars and live and dead fetuses were recorded. The fetuses were weighed, examined for external abnormalities and sexed by internal examination of the gonads. They were subjected to a detailed soft tissue and skeletal examinations.

Ratinale for dose level selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of the following previous studies:

. Dose range-finding study by the oral route (gavage) in non-pregnant rabbits (Citoxlab France/Study No. 44827 TSL):
In this study, three non-mated females received the test item over three consecutive periods of 7-day each and at increasing dose levels (150, 210 and 350 mg/kg/day, respectively). The 150 and 210 mg/kg/day dose levels were administered at a volume of 6 mL/kg and, due to formulation and stability issues, the 350 mg/kg/day dose level at 10 mL/kg/day. The high-dose formulation resulted in a 35 mg/mL formulation which corresponds to the maximum feasible formulation in term of homogeneity and stability. Each female was examined daily for clinical signs. Body weight and food consumption were recorded at designated intervals. At the end of the treatment period, the females were euthanized and submitted to a post-mortem examination of the principal thoracic and abdominal organs with a special attention paid to the stomach. There were no significant findings at any dose levels tested. No Maximal Tolerated Dose (MTD) was achieved. However taking into account the maximum feasible homogeneity and stability of the test item (35 mg/mL) and the maximum recommended volume to be administered in pregnant females (10 mL/kg/day), 350 mg/kg/day could be selected as the highest dose to be tested in a further preliminary study.

. Preliminary study of prenatal developmental toxicity by oral route (gavage) in rabbits (Citoxlab France/Study No. 44828 RSL):
In this study, five mated females received the test item from Days 6 to 28 p.c. inclusive at 40, 120 or 350 mg/kg/day. The dose levels were administered at a volume of 10 mL/kg. Each female was examined daily for clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., animals were sacrificed and submitted to a macroscopic post-mortem examination. A hysterectomy was performed and the numbers of corpora lutea, implantations, uterine scars, early and late resorptions and live and dead fetuses were recorded. The fetuses were weighed, sexed and subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. All fetuses were then discarded. When compared with controls, 350 mg/kg/day was associated with excessive maternal toxicity (clinical signs, body weight loss and decreased food consumption) and low number of live fetuses.

Therefore, 250 mg/kg/day was selected as the high-dose level for this present study. The low-dose and mid-dose were selected using a ratio representing approximately a 2.5-fold interval (i.e. 100 and 40 mg/kg/day).
Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, the animals were observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time of day, for the recording of clinical signs (including evidence of abortion).

BODY WEIGHT: Yes
- Time schedule for examinations:The body weight of each female was recorded on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
- Time schedule for examinations: The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 p.c.
females were euthanized by an intravenous injection of sodium pentobarbital and were submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy. A gross evaluation of placentas was also undertaken.
- Organs examined: liver, stomach, bladder, gonads, uterus.
- Preservation of tissues: Macroscopic lesions observed in females were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative). As remarkable macroscopic lesions were observed in test item-treated group animals, the corresponding tissues of five control animals were sampled and preserved.
- At necropsies, the stomachs of all animals were preserved for a potential further microscopic examination (not performed).


OTHER:
- 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 treatment period, including weekends and public holidays.


Ovaries and uterine content:
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
The ovaries and uterine content was examined after termination: Yes


The ovaries and uterus 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 degenerative changes,
- dead fetus: dead fetus with no degenerative changes.

Gravid uterine weight:
- net body weight change: Body weight on Day 29 p.c. - body weight on Day 6 p.c. - gravid uterine weight

A gross evaluation of placentas was also undertaken.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter or 50% per litter (see details below)]
- Head and brain examinations: Yes: [50% per litter ]


- Body weight of fetuses: The body weight of each fetus was recorded.
- Sex of fetuses: The sex of each fetus was determined by internal examination of the sexual organs at the time of the detailed dissection.


- Examination of the fetuses:
The fetuses were euthanized by intraperitoneal injection of sodium pentobarbital. The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations (Makris et al., 2009, 2011 and Chahoud et al., 1999):
- malformation refers to a permanent structural change that is likely to adversely affect the survival or health,
- variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect the survival or health (this might include a delay in growth or morphogenesis that otherwise followed a normal pattern of development).

- 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:
Body: As soon as possible after euthanasia, all fetuses in each litter were subjected to a detailed dissection of the soft tissue, which included observation of all the organs and structures of the neck, thorax and abdomen. The fetuses were then eviscerated.
Head and brain: One half of the fetuses were decapitated and the head was fixed in Harrison’s fluid with decalcification. Serial sectioning was performed for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison’s fluid. Serial section was made for examination of the organ.

- Skeletal examination: The carcasses of the fetuses were 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 observation of all the bone and cartilage structures of the skull (50% of fetuses), spine, rib cage, pelvis and limbs (100% of fetuses).


Statistics:
Data were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous) or by Fisher’s exact probability test (proportions).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 and 40 mg/kg/day, there were no test item treatment-related clinical signs. At 250 mg/kg/day and when compared with controls, brown coloured urines were observed in most females. This finding was considered to be test item treatment-related but considered as non-adverse.
Reddish vaginal discharge, blood in the bedding, short/broken teeth, difficulty during gavage administration, abnormal growth of teeth (cut regularly) were observed at low incidences in test item-treated and/or control groups. They are findings commonly observed in this species and strain of animals.

Refer to the table 1 in the field below ‘Any other information on results incl. tables' and to the attached document (DOC 1) in the filed below ‘Attached background material’.
.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.

Refer to the attached document (DOC 1) in the field below ‘Attached background material’.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no significant effects on mean body weight.

At 250 mg/kg/day, there was a body weight loss (-18 g vs. +38 g in controls, p<0.001) on Days 6-9 p.c. which returned toward control values thereafter but which did not fully recover (+230 g vs. +302 g in controls on Days 6-29 p.c., not statistically significant). This finding was considered to be test item treatment-related and adverse with minimal importance.

At 100 mg/kg/day and when compared with controls, there was a lower mean body weight gain (+23 g vs. +38 g on Days 6-9 p.c., not statistically significant) followed by higher mean body weight gain (+53 g vs. +24 g on Days 9-12 p.c., p<0.01) with a return toward control values with full recovery thereafter (+305 g vs. +302 g in controls on Days 6-29 p.c.). This finding was considered to be test item treatment-related but not adverse.

At 40 mg/kg/day there were no effects on mean body weight change.

Refer to the table 2 in the field below ‘Any other information on results incl. tables'.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg/day and when compared with controls, there was a lower mean food consumption (-25% vs. controls, p<0.001 on Days 6-9 p.c. and -13% vs. controls on Days 9-12 p.c., p<0.05) which returned toward control values thereafter. This finding was considered to be test item treatment-related and adverse with moderate importance.

At 100 and 40 mg/kg/day and when compared with controls, there were no effects on mean food consumption.

Refer to the table 3 in the field below ‘Any other information on results incl. tables’.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 2/24 females had colored focus(i) on the stomach mucosa.
As this finding was not observed in control animals, a test item treatment-related effect cannot be excluded.

In the 100 and 40 mg/kg/day groups and when compared with controls, there were no test item treatment related findings in animals sacrificed as scheduled, on Day 29 p.c. The few findings recorded are commonly observed findings in this species and strain.

Refer to the attached document (DOC 2) in the field below ‘Attached background material’.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss, early and late resorptions) whatever the administered doses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss, early and late resorptions) whatever the administered doses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss, early and late resorptions) whatever the administered doses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss, early and late resorptions) whatever the administered doses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects on dead fetuses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At hysterectomy on Day 29 p.c., there were 24/24, 22/24, 23/24 and 22/24 pregnant females (with live fetuses) in control, 40, 100 and 250 mg/kg/day groups, respectively.
In low-, mid- and high-dose groups there were two, one and two females which failed to become pregnant while mated.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Gravid uterus and carcass weights:
At 250 mg/kg/day and when compared with controls, there was a low mean gravid uterus weight (-14% vs. controls, p<0.05). This finding was considered to be test item treatment-related but not adverse (no significant effects on mean number of fetuses and mean fetal body weight).
At 100 and 40 mg/kg/day and when compared with controls, there were no effects on mean gravid uterus weights, carcass weights and net body weight changes.

Refer to the table 5 in the field below ‘Any other information on results incl. tables’.

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no significant effects on mean fetal body weight.
At 250 mg/kg/day and when compared with controls, there was a lower non-adverse fetal body weight (associated with the low gravid uterus weight observed at this dose level).

Refer to the table 6 in the field below ‘Any other information on results incl. tables’.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no significant effects on live fetuses whatever the administered doses.

Refer to the table 4 in the field below ‘Any other information on results incl. tables’.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no significant effects on sex ratio (percentage of male foetuses) whatever the administered doses.

Refer to the table 6 in the field below ‘Any other information on results incl. tables’.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no significant effects on mean mumber of live fetuses and no significant effects on mean fetal body weight.

Refer to the tables 4 and 6 in the field below ‘Any other information on results incl. tables’.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item treatment-related external malformations.

Omphalocele, gastroschisis and/or short tail were observed with no dose level relationship (in low-dose and/or control groups, not in high-/mid-dose groups). They are commonly observed fetal external malformations in this species and strain.

Refer to the attached document (DOC 3) in the field below ‘Attached background material’.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item treatment-related skeletal malformations.

There were a few malformations, recorder both in the controls and/or test item-treaded groups for which a test item treatment effect was considered unlikely in the absence of any dose level relationship and/or because of incidence(s) close to the upper limit of the Historical Control Data.

Refer to the table 7 in the field below ‘Any other information on results incl. tables’ and to the attached document (DOC 4) in the field below 'Attached background material'.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Soft tissues examination:

Soft tisue variations:
When compared with controls, there were no statistically significant differences in the distribution of litter and fetal incidences of soft tissues variations whatever the administered doses.
A series of soft tissue variations of the gall bladder, liver, kidneys, gonads, great blood vessels of the heart, abdominal wall and/or abdomen were observed with no dose level relationship (non-dose related distribution and fetal/litter incidence across groups). They are commonly observed fetal soft tissue variations in this species and strain.

Soft tissue malformations:
There were no test item treatment-related soft tissue malformations.
A dilated aortic arch was observed with no dose level relationship (one fetus in the low-dose group). This is a commonly observed fetal soft tissue malformation in this species and strain.


Refer to the attached document (DOC 5) in the field below ‘Attached background material’.
Details on embryotoxic / teratogenic effects:
The other available data are the following:

Fetal external examination
Variations
There were no test item treatment-related external variations.
Polyhydramnios (excess of amniotic fluid in the amniotic sac), abnormal color of amniotic fluid, paw hyperflexion and enlarged abdomen were observed with no dose level relationship (in mid-/low-dose and/or control groups, not in the high-dose group). They are commonly observed fetal external variations in this species and strain.
Refer to the attached document (DOC 6) in the field below ‘Attached back ground material’.


Skeletal examination
Cartilages
In all test item-treated group and when compared with controls, there were higher number of fetuses with non ossified hyoid (cartilage present but not ossified). This observation correlates with the higher incidence of incomplete ossification of the hyoid centrum (see the following § Variations). This finding was considered test item treatment-related (none observed in controls) but not adverse (bone cartilage present).

At examination of the fetal cartilage and when compared with control, there were a few other differences (cartilage of sternebrae(e) or rib(s) present) for which a test item treatment effect was considered unlikely in the absence of any dose level relationship.

Refer to the table 8 in the field below ‘Any other information on results incl. tables’ and to the attached document (DOC 4) in the field below 'Attached back grouund material'.

Variations
In all test item-treated group and when compared with controls, there were higher number of fetuses with incomplete ossification of the hyoid centrum (cartilage present but not ossified , see previous § Cartilages). This finding was considered test item treatment-related (none observed in controls) but not adverse (bone cartilage present) and remained within acceptable ranges when compared with Historical Control Data.

When compared with control, there were a few other statistically significant differences (unossified 5th sternebrae(e), supernumerary 13th ribs(s) and unossified talus) for which a test item treatment effect was considered unlikely in the absence of any dose level relationship and/or because of incidence(s) close to the upper limit of the Historical Control Data.

Refer to the table 9 in the field below ‘Any other information on results incl. tables and to the attached document (DOC 4) in the field below ‘Attached background material’.

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effect on fetus
Developmental effects observed:
no

Tables - Maternal toxicity/effects:

Table 1: Number of females with clinical signs (before and after treatment):

Dose level

(mg/kg/day)

0

40

100

250

Number of females at termination

 

 

 

 

. brown coloured urine

1 (GD 28)

 

 

23

(from GD 13 up to 28)

. reddish vaginal discharge

 

2

(GD 21, 25 and 27)

 

1

(GD 26-27)

. blood in the bedding

 

2

(GD 22-24, 25 and 27)

 

2

(from GD 26 up to 27)

. short/broken teeth

 

1

(GD 6-28)

1

(GD 6-23)

 

. difficulty during gavage administration

2 (GD 9 or 19)

 

2

(GD 20 or 26)

1

(GD 19)

. abnormal growth of teeth

(cut regularly)

 

 

1

(GD 24-28)

 

No remarkable findings

21

21

21

1

GD: Gestation Day (GD0 = Day 0p.c.).

Table 2: Mean body weight and body weight change (g/animal)

Dose level
(mg/kg/day)

0

40

100

250

Body weight (g)

 

 

 

 

Day 6 p.c.

3622

3666

3687

3654

Day 9 p.c.

3660

3705

3709

3636

Day 12 p.c.

3684

3747

3762

3676

Day 15 p.c.

3769

3821

3840

3735

Day 19 p.c.

3819

3879

3891

3799

Day 24 p.c.

3893

3968

3966

3878

Day 29 p.c.

3924

4027

3992

3884

Body weight change (g)

 

 

 

 

Days 6 -9 p.c.

+38

+39

+23

-18#

Days 9-12 p.c.

+24

+42

+53**

+40

Days 12-15 p.c.

+84

+74

+78

+58

Days 15-19 p.c.

+50

+58

+51

+65

Days 19-24 p.c.

+74

+89

+75

+79

Days 24-29 p.c.

+31

+59

+26

+6

Days 6-29 p.c.

+302

+361

+305

+230

Statistical significance: **: p<0.01 and #: p < 0.001.

Table 3: Mean food consumption (g/animal/day)

Dose level
(mg/kg/day)

0

40

100

250

Days 6-9 p.c.

187

174

(-7)

178

(-5)

141#

(-25)

Days 9-12 p.c.

179

177

(-1)

182

(+2)

156*

(-13)

Days 12-15 p.c.

154

148

(-4)

163

(+6)

136

(-12)

Days 15-19 p.c.

173

178

(+3)

176

(+2)

162

(-6)

Days 19-24 p.c.

147

156

(+6)

159

(+8)

145

(-1)

Days 24-29 p.c.

101

117

(+16)

103

(+2)

105

(+4)

Table 4: Hysterectomy data

Dose level
(mg/kg/day)

0

40

100

250

HCD

Number of females with live fetuses

at termination

24

22

23

22

147

Mean number of corpora lutea per animal (± SD)

11.7 ± 1.9

11.2 ± 1.7

11.6 ± 1.5

10.6 ± 2.2

[10.5; 11. 3]

Mean number of implantations per animal (± SD)

10.0 ± 2.4

10.2 ± 1.9

10.3 ± 2.1

9.1 ± 2.4

[8.3; 10.4]

Mean pre-implantation loss (%)

14.9

9.1

11.2

14.4

[7.6; 24.1]

Mean number of live fetuses

per animal (± SD)

9.0 ± 2.2

9.0 ± 2.1

9.3 ± 2.4

8.4 ± 2.5

[7.7; 9.9]

Dead fetuses (%)

0.0

0.0

0.0

0.0

[0.00; 0.21]

Mean number of implantation scars

0.0

0.0

0.0

0.0

/

Mean number of early resorptions

0.4

0.4

0.7

0.6

/

Mean number of late resorptions

0.6

0.7

0.3

0.1

/

Mean post-implantation loss (%)

9.7

10.9

10.4

7.7

[0.5;9.4]

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2013 to November 2014).

[ ]: range of study means (min and max).

/: not recorded in HCD.

Table 5: Net body weight change (g)

Dose level
(mg/kg/day)

0

40

100

250

Gravid uterus weight (g)

496.8

498.5

(0)

510.9

(+3)

425.7*

(-14)

Carcass weight (g)

3426.8

3528.5

(+3)

3481.1

(+2)

3458.4

(+1)

Net body weight change from Day 6p.c.(g)

-194.9

-137.4

-205.7

-195.2

Tables - Effects on fetuses:

Table 6: Mean fetal body weights (g) and mean percentages of male fetuses (%)

 

Dose level
(mg/kg/day)

0

40

100

250

Mean fetal body weight (g ± SD)

37.7 ± 4.9

36.2 ± 4.4

(-4)

36.0 ± 4.3

(-5)

34.6 ± 6.2

(-8)

Mean percentage of male fetuses (%)

55.6

48.8

49.4

51.2

( ): in brackets: percentage differencesvs.control group (%).

Table 7: Litter (L) and Fetal (F) incidences of skeletal malformations

 

Dose level
(mg/kg/day)

0

40

100

250

HCD

Dams with live fetuses, n

24

22

23

22

143

Live fetuses, n

216

199

213

184

1314

. interparietal: absent, L(F) %

12.5 (1.4)

13.6 (2.0)

4.3 (0.5)

22.7 (3.3)

16.7 (2.2) (a)

. parietal: split, L(F) %

0.0 (0.0)

0.0 (0.0)

13.0 (1.9)

0.0 (0.0)

0.0 (0.0) (a)

. skullcap: bone fused, L(F) %

0.0 (0.0)

0.0 (0.0)

4.3 (0.5)

0.0 (0.0)

5.6 (0.6) (a)

. thoracic vertebra(e): fused
arch, L(F) %

0.0 (0.0)

4.5 (0.5)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0) (a)

. caudal vertebra(e): absent, L(F) %

4.2 (0.5)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0) (a)

. fused sternebrae(e), L(F) %

12.5 (1.4)

18.2 (3.0)

21.7 (2.3)

4.5 (1.1)

31.3 (5.1) (a)

. split sternebrae(e), L(F) %

4.2 (0.5)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0)

0.0 (0.0) (a)

. fused ribs, L(F) %

0.0 (0.0)

4.5 (0.5)

0.0 (0.0)

0.0 (0.0)

12.5 (1.3) (a)

. Litters affected, n (%)

7 (29.2)

8 (36.4)

8 (34.8)

6 (27.3)

42 (29.4) (b)

. Fetus affected, n (%)

8 (3.7)

11 (5.5)

10 (4.7)

8 (4.3)

54 (4.1) (b)

n: number.

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2013 to November 2014).

(a): upper limit by study (n = 7 studies).

(b): mean number (percentage) of litters or fetuses affected.

Table 8: Litter (L) and Fetal (F) incidences of cartilage findings

 

Dose level
(mg/kg/day)

0

40

100

250

HCD

Dams with live fetuses, n

24

22

23

23

143

Live fetuses, n

216

199

213

184

1314

. hyoid: cartilage of centrum present, L (F) %

0.0 (0.0)

13.6 (4.0**)

21.7* (3.8**)

13.6 (2.7*)

17.4 (1.9)(a)

. sternebrae: cartilage present, L (F) %

87.5 (45.4)

95.5 (47.2)

95.7 (57.3*)

72.7 (36.4)

87.0 ([DB1] 30.6)(a)

. rib(s): cartilage present, L (F) %

4.2 (0.5)

13.6 (3.5*)

13.0 (2.3)

9.1 (1.1)

4.3 (1.0)(a)

n: number.

 

Table 9: Litter (L) and Fetal (F) incidences of skeletal variations

 

Dose level
(mg/kg/day)

0

40

100

250

HCD

Dams with live fetuses, n

24

22

23

22

143

Live fetuses, n

216

199

213

184

1314

. hyoid: incomplete ossification of centrum, L (F) % (c)

0.0 (0.0)

13.6 (2.5*)

21.7* (3.8**)

13.6 (2.7*)

17.4 (2.7) (a)

. sternebrae: unossified 5thsternebrae, L (F) % (c)

37.5 (8.3)

40.9 (11.1)

73.9* (16.4*)

27.3 (5.4)

43.5 (8.6) (a)

. supernumerary 13thrib(s), L (F) % (c)

100.0 (57.9)

100.0 (60.8)

95.7 (55.9)

100.0 (78.3#)

100 (73.5) (a)

. hindlimb(s): unossified talus, L (F) % (c)

0.0 (0.0)

13.6 (3.0*)

4.3 (0.5)

0.0 (0.0)

6.3 (0.9) (a)

 

Litters affected, n (%) (d)

24 (100.0)

22 (100.0)

23 (100.0)

22 (100.0)

143 (100) (b)

Fetus affected, n (%) (d)

184 (85.2)

180 (90.5)

196* (92.0)

170* (92.4)

1150 (87.5) (b)

n: number.

HCD: Historical Control Data (NZW Rabbits, Centre LAGO France, June 2013 to November 2014).

(a): upper limit by study (n = 7 studies).

(b): mean number (percentage) of litters or fetuses affected.

(c): statistical significance (in terms of numbers of litters or fetuses concerned for individual findings): *: p<0.05; **: p<0.01, #: p < 0.001.

(d): all variations combined (with statistical significance or not).

 

Conclusions:
Based on this study the maternal toxicity observed were a minimal body weight gain loss and a moderate food consumption at the higher tested dose (250 mg/kg/day).
There is an absence of adverse effect whatever the tested dose regarding embryo-fetal development.
Executive summary:

Three groups of 24 mated female New Zealand White rabbits received the test item, daily from gestation Day 6 to Day 28 p.c., by gavage, at dose levels of 40, 100 or 250 mg/kg/day. Another group of 24 mated females received the vehicle (drinking water treated by reverse osmosis), under the same experimental conditions and acted as a control group.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., females were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, uterine scars and live and dead fetuses were recorded. The fetuses were weighed, examined for external abnormalities and sexed by internal examination of the gonads. They were subjected to a detailed soft tissue and skeletal examinations.

At hysterectomy on Day 29 p.c., there were 24/24, 22/24, 23/24 and 22/24 pregnant females (with live fetuses) in control, 40, 100 and 250 mg/kg/day groups, respectively.

At 250 mg/kg/day and when compared with controls, brown coloured urine was observed in most females and was considered to be test item treatment-related, but as non-adverse. At 100 and 40 mg/kg/day, there were no test item treatment-related clinical signs. There were no significant effects on mean boby weight whatever the dose tested. At 250 mg/kg/day, there was a decrease boby weight gain (body weight loss on Days 6 -9 p.c) which tended to return toward control values thereafter. This finding was considered to be test item treatment-related and adverse with minimal importance. At 100 mg/kg/day, there was a low body weight gain followed with a return toward control values. This finding was considered to be test item treatment-related but not adverse. At 40 mg/kg/day there were no effects on mean body weight change. At 250 mg/kg/day, there was a low food consumption on Days 6-12 p.c. with a return toward control values thereafter. This finding was considered to be test item treatment-related and adverse with moderate importance. At 100 and 40 mg/kg/day, there were no effects on mean food consumption. At 250 mg/kg/day and when compared with controls, there was a non-adverse lower mean gravid uterus weight. At 100 and 40 mg/kg/day and when compared with controls, there were no effects on mean gravid uterus weights, carcass weights and net body weight changes. There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites, pre-implantation loss, live fetuses and post-implantation loss) whatever the administered doses. There were no adverse effects on mean fetal body weight, mean placental weight and sex ratio (percentage of male fetuses) whatever the administered doses. There were no test item treatment-related variations or malformations at external and soft tissue examination of the fetuses whatever the administered doses. In all test item-treated group and when compared with controls, there were higher number of fetuses with non or incompletely ossified hyoid (cartilage present but not ossified). This finding was considered test item treatment-related (non observed in controls) but not adverse (bone cartilage present). There were no test item treatment-related skeletal malformations whatever the administered doses.

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 mild maternal toxicity (minimal body weight gain loss and moderate food consumption) at 250 mg/kg/day.

- The No Observed Adverse Effect Level (NOAEL) for embryo-fetal development was considered to be 250 mg/kg/day, based on absence of adverse effect at this dose level. 

      

   

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2017 to 13 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 443
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Mequinol (paramethoxyphenol)
- EC N°: 205-769-8
- CAS N°: 150-76-5
- Description: White flakes
- Purity: higher than 99%.

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
See the same study in the section 'Toxicity to reproduction'.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
See the same study in the section 'Toxicity to reproduction'.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See the same study in the section 'Toxicity to reproduction'.
Details on mating procedure:
See the same study in the section 'Toxicity to reproduction'.
Duration of treatment / exposure:
See the same study in the section 'Toxicity to reproduction'.
Frequency of treatment:
Daily
Duration of test:
See the same study in the section 'Toxicity to reproduction'.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
P generation: 24 rats per sex and per dose.
F1 generation: 20 rats per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
See the same study in the section 'Toxicity to reproduction'.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part as routine examinations. From the start of the treatment period each animal was observed at least twice a day (before and after treatment), at approximately the same time of day, for the recording of clinical signs.
Detailed clinical examinations were performed on all animals once a week until the end of the study.
Observations included (but were not limited to) changes in the 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, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on the first day of treatment (Study Day 1), then at least once a week until mated (or until euthanasia for females with no evidence of mating), on Days 0, 4, 7, 10, 14, 17 and 20 p.c. and, on Days 1, 4, 7, 14 and 21 p.p. Animals were weighed at euthanasia as normal procedure before pathology examination.


OTHER:
Medical care: On a few occasion several animal from all groups received topical application of Cothivet® (essential oils) and/or Antisept® (chlorhexidine). These healing and antiseptic sprays were used to treat wounds/scabs .

Morbidity and mortality: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.
Attention was paid to humane end-points defined in Citoxlab France quality document LI SES 0004. End points specific to the species used in the study were available in the animal facility.
Animals showing signs of poor clinical condition were weighed and humanely euthanized.

Food consumption: The quantity of food consumed by each female was measured at least once a week from the first day of treatment until the start of the mating period, during gestation for the intervals: Days 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 p.c. and during lactation for the intervals: Days 1-4, 4-7, 7-14, and 14-21 p.p..
During the mating period, food consumption was not measured.

OESTROUS CYCLE:
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning:
- during the last 2 weeks of the premating period,
- during the mating period, until the females were mated or the mating period had ended,
A fresh vaginal lavage was done on the day of sacrifice (premature or scheduled).

SACRIFICE:
On completion of the treatment period, all surviving P animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination:
P females: on Days 22-23 p.p.,
P females which did not deliver: on Days 24-26 p.c. (after body weight recording to check for a possible unnoticed delivery),
P female with no evidence of mating: 25 days after the end of the mating period since no delivery occurred,
P females with total litter loss: as appropriate.


Organ weights: The body weight of each animal euthanized as scheduled was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

Macroscopic post-mortem examination: A complete macroscopic post-mortem examination was performed on all P. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of implantation sites were recorded for females euthanized on Days 22-23 p.p. for P generation.

Preservation of tissues: The tissues specified in Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes with optic nerves, testes and epididymides which were preserved in Modified Davidson's Fixative. Ovaries (all groups from P generations) were fixed for at most 96 hours in formalin before being embedded in paraffin wax.

Preparation of histological slides: All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS and for the right ovaries which were stained by PCNA immunohistochemistry). Five step-sections (100 µm apart in the middle third) were made for the right ovary. This tissue processing was performed at Citoxlab France.

Microscopic examination: A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from animals of the control and high-dose groups (groups 1 and 4) euthanized at the end of the treatment period (see table in the section 'Any other information on materials and methods incl. tables)',
- reproductive organs from animals that did not mate or conceive, from pregnant females that did not deliver, from females with abnormal oestrous cycles and from any male with abnormalities at sperm analysis, to investigate possible causes (low- and mid-dose groups),
- all macroscopic lesions of all groups (from P), including those from prematurely euthanized animals.

Right ovary (groups 1 and 4): a detailed and careful microscopic examination was made of five step sections of the right ovary of each P and cohort 1A female, with enumeration of the total number of primordial follicles on PCNA-stained slides.

Based upon the results of the microscopic examination of the high-dose group and after the agreement of the Sponsor, other tissues of the low- and intermediate-dose groups have been examined:
- Microscopic examination on hematoxylin-eosin stained slides :
. forestomach from low- and mid-dose groups of P-Generation (males and females) animals,
. bone marrow (sternum) from low- and mid-dose groups of P-Generation (males and females) animals.
- Enumeration of the total number of primordial follicles on PCNA-stained slides:



Ovaries and uterine content:
See section above
Fetal examinations:
Each live pup was identified individually on Day 1 p.p., by subcutaneous injection of Indian ink.

Litter size:
The total litter size and sex of each pup were recorded on Day 1 p.p.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

Litter size adjustment:
On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible five males and five females per litter. Whenever necessary, partial adjustment (for example six males and four females) was permitted.
Standardization of litter size is considered to reduce the litter size-induced variability in the growth and development of pups and thus increase the sensitivity of statistical analysis. This also ensures that any adverse effects on pup growth and development are not masked by a treatment-related reduction in litter size.

Clinical signs:
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities (including oral cavity and orifices).
The first clinical examination (on Day 1 p.p.) included:
gross external examination (e.g. external visible abnormalities, cleft palate, subcutaneous hemorrhages, abnormal skin color or texture; presence of umbilical cord, lack of milk in the stomach, presence of dried secretion),
qualitative assessment of body temperature, activity and reaction to handling.

Body weight:
The weight of each live pup was recorded on Days 1, 4, 7, 14 and 21 of p.p.

Pup development:
The following physical development measurements were performed in pups of each litter:
anogenital distance (AGD) on Day 4 p.p. (all pups before culling),
number of nipples and of areolae in male pups: on Day 13 p.p.
The AGD was normalized to the cube root of body weight recorded on Day 4 p.p.

Terminal examination:
Pups prematurely euthanized because of dying mother were deeply anesthetized by an intraperitoneal injection of Euthasol (see § Study plan adherence). A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead (except cannibalized) and prematurely euthanized pups. Special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach). Macroscopic lesions were preserved in appropriate fixative.

Indices:
The following parameters were calculated: live birth index, vialibility index on Day 4 p.p., lactation index as indicated below:

- live birth index:
Number of live pups on Day 1 p.p.
_____________________ x 100
Number of delivered pups

- viability index on Day 4 p.p.:
Number of surviving pups on Day 4 p.p. (before culling)
_______________________________________ x 100
Number of delivered pups

- lactation index:
Number of surviving pups on Day 21 p.p.
________________________________________ x 100
Number of surviving pups on Day 4 p.p. (after culling)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Data on females P generation were only reported in this section:

At 250 mg/kg/day, half-closed eyes was recorded in four female rats on the Days 70-73 of the treatment period and was considered to be test item treatment-related. This finding was observed at low incidence, transitory (observed for 1 to 2 hours) after dosing and was not correlated with macroscopic or microscopic (including brain histomorphology examination) findings. Staggering gate/tonic seizures were observed in only one female on Day 99, therefore a test item treatment relationship was considered unlikely.
From 100 mg/kg/day, hypoactivity was observed in almost all treated female rats and was considered to be test item treatment-related. This finding was observed 1 to 2 hours after dosing and was not correlated with macroscopic and microscopic (including brain histomorphology examination) findings.
Ptyalism was observed from 40 mg/kg/day at increased dose level incidences and was considered to be test item treatment-related but not adverse as it is finding commonly observed after repeated oral (gavage) administration in rats.

A series of additional clinical signs were observed in females without any dose level relationship (i.e. abnormal growth of teeth (cut regularly), hunched posture, piloerection, loud breathing, alopecia/thinning of hair, chromodacryorrhea, chromorhinorrhea, increase in size (mammary gland, urogenital region), liquid discharge (nose), pallor (extremities, eye(s)), reflux at dosing, scabs/wound, soiled (urogenital area, neck), thin appearance and/or vaginal discharge). They are commonly observed findings in this species and strain of animals housed in these experimental conditions.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The unscheduled deaths recorded in the P generation females is the following:
F-L24413 (female, control group): with pallor and piloerection from Study Day 96, and apparent difficulties to deliver on Study Day 97 (eight pups alive in the litter) was prematurely sacrificed for human reasons. At necropsy, there were three dead fetuses with placenta and nine placentas in the left uterine horn .
F-L24427 (female, control group): the plastic gavage tube was broken on Study Day 54 during the gavage procedure and swallowed; the female was prematurely sacrificed for human reasons. At necropsy there was a piece of the gavage tube into the stomach.
F-L24471 (female, 100 mg/kg/day): with hunched posture, pallor of eyes and extremities, piloerection, dyspnea, vaginal discharge and dead litter on Study Day 99 was prematurely sacrificed for human reasons. At necropsy, this female had tan discoloration of lungs/bronchi, liver and kidneys, gelatinous thymus and a dead fetus in the vagina.
F-L24476 (female, 100 mg/kg/day): with hypoactivity, lateral recumbency, tonic seizures, hypotonia and dyspnea on Study Day 49 was prematurely sacrificed for human reasons. At necropsy, there was an enlarged spleen. A technical issue at gavage was considered to be the most probable cause of death.
F-L24488 (female, 250 mg/kg/day): with hunched posture, pallor of extremities, piloerection from Day 99 and, hypoactivity regularly from Day 1, and dead litter on Study Day 99 was prematurely sacrificed for human reasons. There were no findings at necropsy.
F-L24491 (female, 250 mg/kg/day): with pallor of extremities, piloerection and cold to the touch on Day 99, hypoactivity regularly from Day 1, and difficulties to deliver on Study Day 99 (two live pups and four dead pups in the litter) was prematurely sacrificed for human reasons. At necropsy, this female had the right uterine horn with nine dead fetuses with placentas and a left uterine horn with eight scars.

Decisions of sacrifice for difficulties to deliver or dead litter were taken for control and/or treated animals. These findings are frequent observations in animalsof this physiological condition and commonly recorded in this species and strain. Therefore a test item treatment-related effect was considered to be unlikely.
Overall, there were no tendencies towards test item treatment-related deaths in male or female animals from the P Generation.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Data on female P generation were only reported in this section:

Premating period (Females):
At 250, 100 and 40 mg/kg/day, when compared with controls, there were no effects on mean body weight and no test item treatment-related effects on meanbody weight change.

Pregnancy period (females):
At 250, 100 and 40 mg/kg/day, when compared with controls, there were no test item treatment-related effects on mean body weight and mean body weight change during the pregnancy period.

Lactation period (females):
Mean body weight: At 250, 100 and 40 mg/kg/day, when compared with controls, there were no test item treatment-related effects on mean body weight during the lactation period.
Mean body weight change: In all test item-treated groups, when compared with controls, there were lower mean body weight changes on Days 1 to 4 p.p. This finding was considered to be test item treatment-related but not adverse based on the return towards control values from Days 4 to 7 p.p.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Data on female P generation were only reported in this section:

Premating (Females):
At 250 mg/kg/day, when compared with controls, there were episodes of lower mean food consumption (down to -13% on Days 36 to 43, p<0.05). This finding was considered to be test item treatment-related and adverse with moderate toxicological importance (less than 20% difference vs. controls with a return towards control values on Days 64 to 71).
At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Pregnancy period (females): At 250, 100 and 40 mg/kg/day, when compared with controls, there were no effects during the pregnancy period.

Lactation period (females): At 250 mg/kg/day, when compared with controls, there was a lower mean food consumption in females (down to -18% on Days 7 to 14 p.p., p<0.01). This finding was considered to be test item treatment-related and to be adverse with moderate toxicological importance (less than 20% difference vs. controls).
At 100 and 40 mg/kg/day, when compared with controls, there were no effects during the lactation period.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Haematological findings:
no effects observed
Description (incidence and severity):
In females P generation: there were no significant effects on hematology and coagulation data.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In female P generation: there were no effects on blood biochemistry parameters.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Urinalysis findings:
no effects observed
Description (incidence and severity):

Female P generation:
There were no effects on urinalysis parameters.
At 40 mg/kg/day, when compared with controls, females had a lower urinary specific gravity (-1%, p<0.05). In the absence of any dose level relationship or a similar finding in males, this observation was considered fortuitous.


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Female P generation: No effect on organ weight.


Refer to the attached documents (DOC 1 and DOC 2) in the field below 'Attached background material’.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):

Female P generation:
Test item-related white masses and/or thickening were noted in the forestomach from 1/22 females (only thickening) treated at 100 mg/kg/day and 2/22 females (only thickening) treated at 250 mg/kg/day. These findings correlated with hyperkeratosis, squamous cell hyperplasia and/or fibrosis of submucosa.

The few other isolated gross observations were considered to be consistent with spontaneous findings encountered in the rats of these strain and age.


Refer to the attached documents (DOC 1 and DOC bis) in the field below 'Attached background material’.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Data on females P generation were only reported in this section:

. Forestomach
Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis were noted in forestomach from some females treated at 250 mg/kg/day. In one female treated at 250 mg/kg/day, it was associated with slight focal ulcer. In a few females treated at 100 mg/kg/day, minimal focal hyperkeratosis and squamous cell hyperplasia were observed. The spectrum of lesions including squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis, and ulcer in one female, was considered to be adverse at 250 mg/kg/day in view of the severities (up to moderate) and of the nature of these lesions (subacute/chronic inflammation).
In view of the very low severity of hyperkeratosis and hyperplasia at 100 mg/kg/day and of the absence of f associated ulceration, these findings were considered to be non-adverse at 100 mg/kg/day.

. Bone marrow
Increased cellularity (with increased myeloid/erythroid ratio) was seen in the bone marrow from females treated at 250 mg/kg/day. It was seen mostly in animals with the forestomach lesions recorded with highest grades. This finding was considered to be probably secondary to the forestomach inflammatory process and was considered to be non-adverse in view of its low magnitude and of the absence of clinical pathology correlates.

The remaining microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature, the findings were distributed randomly among groups, and/or their appearance was similar to changes found in controls.
This included, among others:
. minimal to moderate crusts and acanthosis in the skin from test item-treated males and females with poor dose-relationship common on the rats kept in laboratory conditions,
. minimal hepatocellular hypertrophy in 2/22 females treated at 250 mg/kg/day,
. minimal or marked suppurative inflammation in the kidneys from 2/22 females treated at 250 mg/kg/day.

There were no test item-related changes in female reproductive organs nor in nervous system.
There was a good correspondence between the vaginal smears and the histopathological examination of estrus cycle.


Refer to the attached documents (DOC 1 and DOC 1ter) in the field below 'Attached background material’.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones: There were no effects on mean plasma thyroid hormones (T4 and TSH) concentrations in P generation animals (females).


Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were no dose-related effects on mean percentage of pre-implantation loss and on percentage of post-implantation loss. Therefore a test-item treatment related effect was considered unlikely.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
At 250 and 100 mg/kg/day, there were the following findings:
- Mean litter size: when compared with controls and while the differences were not statistically significant, there was an apparent dose-related decrease with values below the lower limit of the Historical Control Data (11.7 and 10.4 vs. 11.8, at 100 and 250 mg/kg/day respectively).
- Number of dead, missing and/or cannibalized pups on Days 1-4 p.p.: when compared with controls, there was a statistically significant increase in the number of dead, missing and/or cannibalized pups. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data.

At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a consequence of lack of maternal care.

When taken together, these finding (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day.

At 40 mg/kg/day, there were no significant effects.

At 250 mg and 100 mg/kg/day, when compared with controls, there was a low number of pups surviving 4 days (with statistically significant differences: p<0.05 and p<0.001, respectively) as a consequence of the low litter sizes at birth and presence of two females with a full dead litter (L24488 and L24471 at 250 and 100 mg/kg/day, respectively). This finding was considered to be adverse and a secondary consequence of lack of maternal care at these dose levels.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index (%) and mean duration of gestation were not affected by the test-item treatment.

Refer to the attached document (DOC 1) in the field below 'Attached background material’.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
When compared with controls or Historical Control Data, there were no test item dose-related effects on number of pregnant females, on mating (mating index), on mating behavior (time taken to mate) and on fertility (fertility index).


Dose level (mg/kg/day) 0 40 100 250 HCD
Number of pregnant females 23 23 19 23 165

Refer also to the attached document (DOC 1) in the field below 'Attached background material’.
Other effects:
no effects observed
Details on maternal toxic effects:
The main maternal toxic effects is neurological clinical signs such as hypoactivity observed from 100 mg/kg/day and half-close eyes noted at 250 mg/kg/day showing mild impact of the substance on the central nervous system (CNS). Inflammation of the forestomach was also observed in females. Dose-related minimal to moderate squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and/or fibrosis were noted in forestomach from some females treated at 250 mg/kg/day. In a few females treated at 100 mg/kg/day, minimal focal hyperkeratosis and squamous cell hyperplasia were observed.(see tables below and also the attached document (DOC 1) in the field below 'Attached background material’.').

Table: Clinical signs in terminated as scheduled P generation females:
(Number of animals affected per group and displaying at least once the clinical sign throughout the study period)


Dose level (mg/kg/day) 0 40 100 250
Number of animals terminated as scheduled 22 24 22 22
Ptyalism 1 2 22 22
(Day 111) (Days 16-75) (Days 6-121) (Days 1-121)
Hypoactivity (episodes, 1 to 2 hours after dosing) 19 22
(Days 72-119) (Days 1-121)
Half-closed eyes (episodes, 1 to 2 hours after dosing) 4
(Days 70-73)
Staggering gate/tonic seizures (1 to 2 hours after dosing) 1
(Day 99)


Table: Histopathology: Main microscopic lesions (incidences) - P generation (females)

Sex Female
Group 1 2 3 4
Dose-level (mg/kg/day) 0 40 100 250
Number of animals per group 22 24 22 22

Forestomach; ulcer
. grade 2 - - - 1
Forestomach; hyperplasia; squamous cell
. grade 1 - - 2 6
. grade 2 - - - 3
. grade 3 - - - 1
Forestomach; hyperkeratosis
. grade 1 - - 3 6
. grade 2 - - - 4
. grade 3 - - - -
Forestomach; hemorrhage
. grade 1 - - - -
Forestomach, infiltrate; mixed cell
. grade 1 - - - -
. grade 2 - - - 2
Forestomach; edema
. grade 1 - - - -
. grade 2 - - - -
. grade 3 - - - 1
Forestomach; fibrosis
. grade 1 - - - -
. grade 2 - - - -
. grade 3 - - - 1
Bone marrow; increased cellularity
. grade 1 - - - 1
. grade 2 - - - 1
Bone marrow; increased myeloid/erythroid ratio
. grade 1 - - - 1
. grade 2 - - - 1

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Clinical signs such as hypoactivity has been observed from 100 mg/kg/day. Inflammation of the forestomach was noted at 250 mg/kg/day and a reduction of the food consumption was observed at 250 mg/kg/day.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
In test item-treated groups and when compared with controls, there were no test item treatment-related effects on pup boby weight.

At 250 mg/kg/day, when compared with controls, there was a low body weight change in female pups on Days 7 to 14 p.p. Taking into account the amplitude of the difference and the return towards control value thereafter, this finding was considered to be not adverse.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg and 100 mg/kg/day, when compared with controls, there was a low number of pups surviving 4 days (with statistically significant differences: p<0.05 and p<0.001, respectively) as a consequence of the low litter sizes at birth (see section 'Dead fetuses' above) and presence of two females with a full dead litter (L24488 and L24471 at 250 and 100 mg/kg/day, respectively). This finding was considered to be adverse and a secondary consequence of lack of maternal care at these dose levels.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
In test item-treated groups and when compared with controls, there were no effects on sex-ratio (percentage of male pups).

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 250 and 100 mg/kg/day, there was a reduction of Mean litter size when compared with controls and while the differences were not statistically significant, there was an apparent dose-related decrease with values below the lower limit of the Historical Control Data of the mean litter size at birth (11.7 and 10.4 vs. 11.8, at 100 and 250 mg/kg/day respectively).

At 250 and 100 mg/kg/day there was a statistically significant increase in the number of dead, missing and/or cannibalized pups on Days 1-4 p.p when compared with controls. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data.

At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a consequence of lack of maternal care.

When taken together, these finding (lack fof maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. These effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day.

At 40 mg/kg/day, there were no significant effects.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
At Day 4 p.p and at the dose level of 250 and 100 mg/kg/day, when compared with controls, there was a low number of pups surviving 4 days (with statistically significant differences: p<0.05 and p<0.001, respectively) as a consequence of the low litter sizes at birth (see previous §) and presence of two females with a full dead litter (L24488 and L24471 at 250 and 100 mg/kg/day, respectively). This finding was considered to be adverse but as a secondary consequence of lack of maternal care at these dose levels (see previous §).

After culling on Day 4 p.p., there were no effects on mean litter sizes.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
External malformations:
no effects observed
Description (incidence and severity):
Clinical observations during lactation:
In test item-treated groups and when compared with controls, there were no pups with malformation(s) or specific test item treatment-related findings at gross external examination, qualitative assessment of body temperature and, activity and reaction to handling in Day 1 p.p. pups.

From 40 mg/kg/day, a few pups had hypoactivity. This finding was transient at 40 and 100 mg/kg/day (observed on one day) and persistent at 250 mg/kg/day (observed for 22 days). Taking into account the occurrence and reversibility of this finding, a test-item treatment-related effect was not excluded at 250 mg/kg/day.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
All other findings recorded in pups during the lactation period are common observations in this species and strain of rats maintained in the experimental condition of this study.

In test item-treated groups and when compared with controls, there were no evidence of any test item treatment-related findings in pups culled on Day 4 p.p. or sacrifice after weaning.
At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day respectively). This finding was considered to be a secondary consequence of lack of maternal care, as previously mentioned.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No adverse effect on mean anogenital distances. There were no areolae or nipples in Day 13 p.p. male pups. There were no effects on sex-ratio. See details on the section 'Toxicity to reproduction'.

Refer to the attached document (DOC 3) in the field below 'Attached background material’.
Details on embryotoxic / teratogenic effects:
The main effects observed were a decrease in mean litter size and an increase in number of dead, missing and/or cannibalized pups.
At 250 and 100 mg/kg/day, there were the following findings:
- mean litter size: when compared with controls and while the differences were not statistically significant, there was an apparent dose-related decrease of mean litter size with values below the lower limit of the Historical Control Data (11.7 and 10.4 vs. 11.8, at 100 and 250 mg/kg/day, respectively),
- number of dead, missing and/or cannibalized pups on Days 1-4 p.p.: when compared with controls, there was a statistically significant increase in the number of dead, missing and/or cannibalized pups. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data.
- number of pups surviving 4 days: when compared with controls, there was a low number of pups surviving 4 days (with statistically significant differences: p<0.05 and p<0.001, respectively) as a consequence of the low litter sizes at birth (see above) and presence of two females with a full dead litter (L24488 and L24471 at 250 and 100 mg/kg/day, respectively).

At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach (13/19 and 9/15 vs. 1/6 in controls, at 100 and 250 mg/kg/day, respectively). This finding was considered to be a consequence of lack of maternal care.
When taken together, these finding (lack of maternal care resulting in decreased mean litter size, increase number of dead, missing and/or cannibalized pups and lower number of pups surviving 4 days) were considered to be test item treatment-related and adverse from 100 mg/kg/day. These effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day.
At 40 mg/kg/day, there were no significant effects.

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in litter size and weights
Remarks on result:
other:
Remarks:
These findings (reduction of litter size and increase in the number of dead, missing and/or cannibalized pups) were not considered as an effect on developement but as a secondary consequence of lack of maternal care. When taken together, lack fo maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day.
Developmental effects observed:
no
Conclusions:
Based on this study the main effects observed in parents and in F1 cohorts from 100 mg/kg/day are clinical signs such as hypoactivity and half-closed eyes showing effects on central nervous system (CNS). Inflammation of the forestomach was also noted in parents and in F1 cohorts (cohorts 1A and 2A). This inflammation was considered as adverse at 250 mg/kg/day in parents and in cohort 2A and from 100 mg/kg/day in cohort 1A for females. The data on parents did not show effect on reproduction/fertility. For P generation offspring, there was an apparent, but not statistically significant, dose-related decrease of mean litter size at the two higher doses and a statistically significant increase, but not dose-related, in the number of dead, missing and/or cannibalized pups at the same doses. At necropsy of pups found dead there were higher numbers of pups with absent of milk in the stomach. This finding was considered to be a consequence of lack of maternal care. When taken together, these finding (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity. Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day. There is an absence of direct developmental effect on pups. Furthermore, no malformations were observed in pups and there is an absence of test item treatment-related adverse findings in cohorts 2A and 2B regarding developmental neurotoxicity.

Executive summary:

In this OECD 443 study, sexually-mature male and female Sprague-Dawley rats (parental (P) generation) (four groups of 24 males and 24 females) were exposed to graduated doses (0, 40, 100 or 250 mg/kg/day) of paramethoxyphenol starting 10 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were selected and assigned to cohorts of animals for:

a) reproductive/developmental toxicity testing: cohort 1A and cohort 1B (without mating to produce a F2 generation),

b) developmental neurotoxicity testing: cohorts 2A and 2B.

 

The F1 offspring received further treatment with the test item (same dose levels than the P generation) from weaning to adulthood or sacrifice. The treatment schedules were the following: daily from weaning (Day 22 p.p.) until euthanasia (from Days 92 to 101 p.p.) for cohorts 1A and 1B and daily from weaning (Day 22 p.p.) until euthanasia (on Days 75 to 78 p.p.) for cohort 2A.

 

Examination:

 

Parents and F1 generation:For parental and F1 generations, clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. P generation males and females were paired until mating was obtained or 14 days had elapsed. Females from the P generation were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. During lactation, the F1 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. When feasible, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter (no culling for litter with less than 10 pups). Pup physical development was assessed at designated end-points.

 

Cohorts:Cohorts 1A and 1B: Animals were selected for assessment of effects upon reproductive systems and of general toxicity. Estrous cycle stages were determined daily for all cohort 1A females, after the onset of vaginal patency, until the first cornified smear was recorded (estrus) and for at least two weeks before the end of the treatment.

Cohort 2A: On Day 22 p.p., 20 pups/group (10 males and 10 females/group; one male or one female/litter; all litters represented by at least one pup; randomly selected) were selected for neurobehavioral testing.

 

Terminal examinations:A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and not selected F1 pups on Day 22 p.p.). This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs. The ratio of organ weight to body weight was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and a complete list of tissues from animals of all or control-/high-dose groups.

 

P generation and cohort 1A: The first 10/sex/group surviving animals were deprived of food for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cells evaluation was performed on surviving males from all or control-/high-dose groups: motility, morphology, sperm cell heads count in testicular/epididymal tissues.

 

Cohorts 2A and 2B: neurohistopathology was performed for all high-dose and control animals of each sex following completion of neurobehavioral testing (between Days 75 and 78 p.p. for cohort 2A animals and on Day 22 p.p. for cohort 2B animals).

 

Thyroid hormones levels (P generation, F1 culled pups and cohort 1A animals): prior to blood sampling the animals were not deprived of food. Blood samples were taken on Day 4 p.p. (F1 culled pups) for measurements of thyroid hormone (T4) levels and, on Day 22 p.p. (F1 pups not selected for cohorts) and at termination from the first ten surviving males/group and the first ten surviving females/group (P and cohort 1A animals), for measurements of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.

 

Results 

P generation 

 

Mortality: there were no test item treatment-related deaths in male or female animals from the P Generation.

 

Clinical signs (males and females):  at 250 mg/kg/day, half-closed eyes recorded in a few animals was considered to be test item treatment-related. From 100 mg/kg/day, hypoactivity recorded in almost all treated rats was considered to be test item treatment-related. From 40 mg/kg/day, ptyalism (a common finding usually observed after repeated gavage administrations) was observed at increased dose level incidences and was considered to be test item treatment-related but non adverse.

 

Mean body weight: males:at 250 mg/kg/day, there was a low mean body weight which was considered to be test item treatment-related and adverse. At 100 and 40 mg/kg/day, there were no effects.

Females:there were no effects during the premating, gestation or lactation periods.

 

Mean body weight change: males: at 250 mg/kg/day, there was a low mean body weight gain which was considered to be test item treatment-related and adverse. At 100 and 40 mg/kg/day, there were no relevant findings.

Females: at 250, 100 and 40 mg/kg/day, there were no relevant findings during the premating, or gestation. In all test item-treated groups, lower mean body weight gains during the lactation period (Days 1 to 4 p.p.) were recorded. These findings were considered to be test item treatment-related but not adverse based on returns towards control values from Days 4 to 7 p.p.

 

Food consumption: males: at 250, 100 and 40 mg/kg/day, there were no effects. Females: during the premating period and at 250 mg/kg/day, there was a low mean food consumption. This finding was considered to be test item treatment-related and adverse but of moderate toxicological importance. During the gestation period, there were no significant effects. During the lactation period and at 250 mg/kg/day, there was a low mean food consumption. This finding was considered to be test item treatment-related and adverse but of moderate toxicological importance. At 100 and 40 mg/kg/day, there were no effects.

 

Estrous cycle: at 250 mg/kg/day, there was a high mean number of days of diestrus and a low mean number of days of proestrus. This finding was considered to be test item treatment-related but not adverse as all values remained within the range of Historical Control Data. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Mating and fertility: there were no test item dose-related effects on mating (mating index), mating behavior (time taken to mate) and fertility (fertility index).

 

Delivery data: there were no test item treatment-related significant effects on mean duration of gestation, number of females with live born, percentage of pre- and post-implantation loss, pup live birth index, and pup sex ratio.

 

Laboratory investigations: there were no relevant effects on hematology and coagulation parameters and on blood biochemistry. There were no test item treatment-related findings on urinalysis parameters. There were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentration. There were no test item treatment-related effects on sperm analysis.

 

 

P generation offspring (pre-weaning F1 pups):

 

Mortality: at 250 and 100 mg/kg/day and when compared with controls, there was a dose-related decrease in mean litter size with values below the lower limit of the Historical Control Data and a statistically significant increase in the number of dead, missing and/or cannibalized pups on Days 1-4 p.p. When balanced with the total number of pups in each group, there were no more significant differences in terms of percentage values. However, the percentages values remained higher than Historical Control Data. At necropsy of pups found dead, there were higher numbers of pups with absent of milk in the stomach. This finding was considered to be a consequence of lack of maternal care. At 40 mg/kg/day, there were no significant effects. When taken together, these findings observed at the two higher doses (lack of maternal care resulting in decreased mean litter size and increase number of dead, missing and/or cannibalized pups) were considered to be test-item treatment related and adverse from 100 mg/kg/day. However, these effects are considered to be indirect via maternal toxicity.  Specifically, the ability of the dam to successfully deliver and care for her offspring was compromised due to the CNS effects (hypoactivity and half closed eyes) of test material at 100 and 250 mg/kg/day. There were no direct effects of the test-item on pup development.

 

Clinical signs: in test item-treated groups and when compared with controls, there were no test item treatment-related significant effects.

 

Body weight: there were no toxicologically significant effects.

 

Anogenital distance (Day 4 p.p.): there was a dose-related decrease in AGD/BW1/3ratio in male pups from 100 mg/kg/day onwards, reaching statistical significance at 250 mg/kg/day. In the absence of effects on mean age at balanopreputial separation (cohorts 1A, 1B and 2A), this finding was considered to be of non-toxicological significance.

 

Areolae and nipples (Day 13 p.p.): there were no areolae or nipples in Day 13 p.p. male pups.

Sex ratio (Day 21 p.p): there were no statistically significant differences.

 

Thyroid hormones: there were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentrations in no-selected pups (Day 4 or 22 p.p.)

 

Macroscopic post-mortem observation: there were no evidence of any test item treatment-related findings in found dead, pups culled on Day 4 p.p. or pups sacrifice after weaning. 

 

Cohort 1A

 

Mortality: there were no test item treatment-related unscheduled deaths.

 

Clinical signs: males:from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were observed in all animals. These findings were considered to be test item treatment-related. Females: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were observed in all animals. At 250 mg/kg/day half-closed eyes were noted in all animals. These findings were considered to be test item treatment-related.

 

Mean body weight: males: at 250 mg/kg/day, there were episodes of lower mean body weight after weaning which were considered to be test item treatment-related and‑adverse. At 100 and 40 mg/kg/day, when compared with control, there were no effects.Females: at 250 mg/kg/day, there was a lower mean body weight after weaning which was considered to be test item treatment-related and-adverse. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Mean body weight change: at 250 mg/kg/day, in both sexes and when compared with controls, there were lower mean body weight gains after weaning. This finding was considered to be test item treatment‑related but non-adverse. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Food consumption: there were no significant effects on male or female mean food consumptions.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 1A animals and no effects on the mean time from vaginal opening to first oestrous in cohort 1A females.

 

Estrous cyles: there were no effects on mean estrous cycle parameters in cohort 1A females.

 

Laboratory investigations: there were no relevant effects on hematology and coagulation parameters and on blood biochemistry. There were no test item treatment-related findings on urinalysis parameters. There were no effects on mean plasma thyroid hormones (T4 and/or TSH) concentration. There were no test item treatment-related effects on sperm analysis.

 

 

Cohort 1B

 

Mortality: there were no unscheduled deaths.

 

Clinical signs: males and females: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were recorded. These findings were considered to be test item treatment-related. At 40 mg/kg/day, there were no test item treatment-related findings.

 

Body weight and body weight change: males: there were no effects on mean body weights or mean body weight changes. Females: there were no relevant effects on mean body weights or mean body weight changes.

 

Food consumption: there were no relevant test item treatment-related effects on mean food consumption.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 1B animals.

 

Cohorts 2A

 

Mortality: there were no unscheduled deaths in cohort 2A males or females.

 

Clinical signs: from 100 mg/kg/day, ptyalism and hypoactivity (for 1 to 2 hours duration after dosing) were recorded. These findings were considered to be test item treatment-related. At 40 mg/kg/day, there were no test item treatment-related findings.

 

Body weight: males: there were no effects on mean body weight. Females: at 250 mg/kg/day, there was a lower mean body weight after weaning. This finding was considered to be test item treatment-related but of minimal toxicological importance [based on the return towards control values from Day 22 (Day 43 p.p.)]. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

Body weight change: at 250 mg/kg/day, in both sexes, there were lower mean body weight gains after weaning. This finding was considered to be test item treatment-related but of minimal toxicological importance [based on the return towards control values from Days 8-15 (Days 29-36p.p.)]. At 100 and 40 mg/kg/day, when compared with controls, there were no effects.

 

Food consumption: there were no effects on mean food consumption in cohort 2A animals.

 

Neurobehavioral testing:

.  Auditory startle test: there were no effects.

.  Functional Observation Battery (FOB): there was no abnormal reactivity to manipulation or to different stimuli,

.  Motor activity: there were no effects on mean number of horizontal movements and rearings.

 

Sexual development: there were no effects on the mean age of balanopreputial separation or mean age of vaginal opening in cohort 2A animals

 

 

Pathology:

 

Parental generation:Decreased thymus weights were recorded in males treated at 250 mg/kg/day with no clear microscopic correlates. Test item-related white masses and/or thickening were noted in the forestomach from one female treated at 100 mg/kg/day and in two males and two females treated at 250 mg/kg/day. Dose-related adverse squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate, edema and fibrosis were noted in males and females treated at 250 mg/kg/day. In one female treated at 250 mg/kg/day, it was associated with slight ulcer. Non-adverse squamous cell hyperplasia and hyperkeratosis were noted in males and females treated at 100 mg/kg/day. Non adverse increased cellularity (with increased myeloid/erythroid ratio) was seen in the bone marrow from males and females treated at 250 mg/kg/day. This finding was considered to be of low toxicological importance.At quantitative evaluation of primordium follicles, there were no test item-related differences between the high-dose and the control groups.

 

Cohort 1A:Decreased thymus weights were recorded in males treated at 250 mg/kg/day. There were no test item-related gross changes. Dose-related adverse squamous cell hyperplasia, hyperkeratosis, hemorrhage, mixed cell infiltrate and edema were noted in the forestomach from males and/or females treated at 250 mg/kg/day, and in females treated at 100 mg/kg/day. In one male treated at 250 mg/kg/day, minimal focal myofiber degeneration was recorded. In one other male, in one female treated at 250 mg/kg/day and in one female treated at 100 mg/kg/day, minimal to slight ulcer was also noted. Non-adverse squamous cell hyperplasia and hyperkeratosis were noted in females treated at 40 mg/kg/day. No changes were noted in the bone marrow.At quantitative evaluation of primordium follicles, there was a minimal decrease in high-dose females when compared to controls. However, a relationship to test item administration was considered to be unlikely in view of the low magnitude of this difference that did not reach statistical significance and of the high inter-individual variability. 

 

Cohort 1B:There were no test item-related organ weight or gross changes. There were no test item-related microscopic findings.

 

Cohort 2A:There were no test item-related organ weight changes. A black discoloration was noted in the forestomach from 1/10 high-dose females that correlated with slight ulcer together with edema, mixed inflammation, hyperkeratosis, acanthosis and hemorrhage. This was related to test item administration although seen at low incidence and severity, in view of the results in the other cohorts and was considered to be adverse. At neuropathology, there were no differences between controls and test item-treated groups for the measurements performed.

 

Cohort 2B:There were no test item-related organ weight or gross changes. There were no test item-related microscopic findings. At neuropathology, there were no differences between controls and test item-treated groups for the measurements performed.

 

Non selected pups:there was no reported mortality. There were not test item-related organ weight differences or gross changes. No microscopic examination was performed.

 

 

Conclusion

  

a) Systemic toxicity evaluation:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity (excluded reproductive and developmental toxicity endpoints) was considered to be 40 mg/kg/day, based on clinical signs from 100 mg/kg/day, adverse histopathological changes in the forestomach from 100 mg/kg/day in females, decreased body weight at 250 mg/kg/day (both in males and females) and adverse histopathological changes in the forestomach at 250 mg/kg/day in males.

 

b) Reproductive/developmental toxicity testing:

The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be 250 mg/kg/day, based on the absence of direct test item treatment-related adverse findings in P generation, cohorts 1A or 1B animals. Decreased mean litter size, increase number of dead, missing and/or cannibalized pups were observed from 100 mg/kg/day and were considered as a secondary consequence of lack of maternal care in F1 offspring on Days 1-4p.p.

 

c) Developmental neurotoxicity testing:

The No Observed Adverse Effect Level (NOAEL) for developmental neurotoxicity was considered to be 250 mg/kg/day based on absence of test item treatment-related adverse findings in cohorts 2A or 2B animals.

 

 

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The conclusion was based on a weight of evidence approach based on three reliable studies: the OECD 414 study on rats performed in 2013, the OECD 414 study on rabbits conducted in 2019 and the OECD 443 study on rats performed on 2019. The reliability of all these studies is 1 under Klimisch scale. The lowest NOAEL has been selected (250 mg/kg/day).
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

Five studies are available. Two with reliability 3 and three with reliability 1.

The two studies scored 3 according to Klimisch scale are briefly described below:

- 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. However since the rats were exposed only at day 11 of gestation no conclusion can be drawn based on this study regarding developmental effect.

- 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

One OECD 414 study on rats was performed in 2013 (CitoxLab, 2013) (Reliability 1 under Klimisch scale). While the study was conducted according to the guideline and under GLP conditions, the high dose level was exceeded the guideline and therefore fetal endpoints at the 400 mg/kg/day dose level are not considered relevant for evaluation in this study. 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 of very severe (excessive) maternal toxicity, 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 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 upon no findings at this dose level. As noted earlier fetal observations at 400 mg/kg/day are not considered relevant for classification and labelling purposes in this study due to the excessive maternal toxicity in this dose level.

 

As indicated in the field 'Description of key information' two other studies have been conducted in 2019 with reliability 1 according to Klimisch scale. An OECD 414 study in rabbits based on which no developmental effect related to the substance was observed in this species and an OECD 443 study conducted on rats. Based on this last study and up to the dose level of 250 mg/kg/day no variations and no malformations have been observed in rats.

Justification for classification or non-classification

Based on the OECD 414 study alone performed in rats in 2013 the substance, paramethoxyphenol has been classified as Reprotoxic Category 2 H361d (suspected of damaging the unborn child), according to the CLP 1272/2008 Regulation. This study showed in fetuse rats an increase in the incidence of variations (external and skeletal) and of malformations (external, soft tissues and skeletal) at the highest tested dose 400 mg/kg/day in a context of very marked/severe maternal toxicity. An increase of post-implantation losses (mainly as a consequence of increased early resorptions) was also observed at 400 mg/kg/day.

In the guidance of the application of the CLP Regulation, it is specified that 'developmental effects occurring even in the presence of maternal toxicity are considered to be evidence of developmental toxicity, unless it can be unequivocally demonstrated on a case-by-case basis that the developmental effects are secondary to maternal toxicity'. Based only on this pre-natal study which was available at the time, there was not enough elements to be certain that the developmental effects observed in pup rats were secondary to the maternal toxicity even if the findings observed in parents were very severe. Therefore, in a conservative approach and due to lack of additional data, the classification Reprotoxic Cat. 2, H361d has been proposed for the substance Paramethoxyphenol.

 

Further investigations were done. According to the compliance checked received from ECHA two additional studies were done: An OECD 414 study on rabbits and an OECD 443 study on rats. No developmental effects were seen in rabbits based on the OECD 414 study performed in 2019 in a second species. The OECD 443 study performed in 2019 in rats showed no test item related variations and malformations on the F1 generation. Furthermore, no effect on brain was noted in the cohorts 2A and 2B. Therefore based on a weight of evidence approach taken into account the 3 reliable studies (the OECD 414 study in rats, the OECD 414 study in rabbits and the OECD 443 study in rats) the substance Paramethoxyphenol is considered to have no impact on the development even in the presence of maternal toxicity. The variations and the malformations observed only in the OECD 414 study performed in 2013 in rats were noted at the highest tested dose (400 mg/kg/day) in a context of very marked and too excessive maternal toxicity. The fact that these variations/malformations have not been observed in the two other reliable studies in the presence of moderate maternal toxicity is an evidence that the variations/malformations noted in the OECD 414 study performed in 2013 in rats is an indirect effect due to the marked and too excessive maternal toxicity at the higher dose, a dose level that should not be considered relevant for classification and labelling purposes.

In a weight of evidence approach taken into account all the reliable reprotoxic/developmental data, it is thus reasonable to assume that the developmental toxicity only seen at the highest tested dose and only in one study on four available is produced solely as a secondary consequence of maternal toxicity and discount the variations/malformations observed in the OECD 414 study in rats. Therefore, the classification of the substance Paramethoxyphenol for developmental effects is not warranted.

 

There is no need to classify the substance Paramethoxyphenol as a reprotoxic (fertility) according to the CLP 1272/2008 Regulation since no adverse effect related to the substance were seen in the two reprotoxic studies, the OECD 422 study (Harlan, 2009) and the OECD 443 study (Citoxlab, 20019b).