Methyl (R)-2-(4-hydroxyphenoxy)propionate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 1999 to 22 April 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 87/302/EEC (or Annex V, 92/69/EEC)

Version / remarks:

18th November 1987

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

The objective of this study was to evaluate the potential toxic effects of the test material upon gonadal function, mating behaviour, conception, development of the concepti during gestation, parturition and development of the pups during lactation, following daily oral administration (gavage) to male and female rats from before mating, through mating, gestation and lactation.
The oral route was selected since it is the route of exposure which is requested by authorities for this type of product.

Test material

Test material information

Constituent 1

Reference substance name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

EC Number:

411-950-4

EC Name:

Methyl (R)-2-(4-hydroxyphenoxy)propionate

Cas Number:

96562-58-2

Molecular formula:

C10H12O4

IUPAC Name:

methyl (2R)-2-(4-hydroxyphenoxy)propanoate

Test material form:

solid: flakes

Details on test material:

- Appearance: White flakes
- Storage conditions of test material: At room temperature and protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl CD® (SD) IGS BR

Details on species / strain selection:

The rat was chosen because it is a rodent species generally accepted by regulatory authorities and the Sprague-Dawley strain was selected because of the availability of background data from previous studies at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sanitary status: Caesarean Obtained, Barrier Sustained-Virus Antibody Free, (COBS-V AF®)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males were 6 weeks old and females were 10 weeks old
- Weight at study initiation: Males had a mean body weight of 187 g (range: 167 to 208 g) and the females 259 g (range: 237 to 287 g)
- Fasting period before study: No
- Housing: Animals were housed individually in polycarbonate cages (43.0 x 21.5 x 20.0 cm) containing autoclaved sawdust, with wood shavings as nesting material, or in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metallic tray containing autoclaved sawdust was placed under the wire-mesh cages. The sawdust was changed at least once a week.
- Diet: ad libitum pelleted diet
- Water: ad libitum access to bottles containing filtered water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 20 % (relative)
- Air changes: About 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h/12 h light/dark cycle (07:00 to 19:00)

IN-LIFE DATES
- From: 07 December 1999 (males) and 01 February 2000
- To: 13 and 15 March 20 (males) and 21 March and 22 April 2000 (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % aqueous methylcellulose solution prepared in water purified by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was administered as a suspension in the vehicle. The test material was ground to fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the required concentrations and then homogenised using a magnetic stirrer. From day 32, the dosage form at 200 mg/mL was mixed using an ultra-turrax laboratory mixer for 3 minutes in order to improve its fluidity and facilitate the gavage procedure. The dosage form preparations were made for up to 9 days of treatment and stored at +4 °C protected from light prior to use. They were delivered each day to the animal room, protected from light and stirred continuously throughout the dosing procedure.

VEHICLE
- Concentration in vehicle: 3, 30 and 200 mg/mL
- Dose volume: 5 mL/kg/day
- Lot/batch no.: methylcellulose batch numbers 118H0286 and 89H0123 supplied by Sigma, France

Details on mating procedure:

- M/F ratio per cage: 1:2; two females were placed with one male, in the latter's cage, during the night.
- Length of cohabitation: Each female was placed with the same male until mating occurred or 21 days had elapsed.
- Proof of pregnancy: Confirmation of mating was made in the morning by checking the presence of a vaginal plug or of sperm in a vaginal lavage. The stage of oestrous cycle was determined at that time, until mating was obtained. The day of confirmed mating was designated day 0 post-coitum.
- After successful mating each pregnant female was caged: Individually
- Other parameters: The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CHEMICAL ANALYSIS OF THE PREPARATIONS
Before the start of treatment, the suitability of the proposed dosage form procedure was determined by the analysis of homogeneity and stability. During the course of treatment, the concentration of the test material in dosage forms prepared for use in the study was checked on a regular basis. The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8, 12 and 16 was determined.

HOMOGENEITY
Before the start of the treatment period, two dosage forms were made, under conditions representative of those of the study: a dosage form at the lowest concentration (3 mg/mL) and a dosage form at the highest concentration (200 mg/mL). Duplicate samples were taken at three different levels (top, middle and bottom) from each dosage form, and analysed for the concentration of the test material to evaluate the homogeneity.
The results of the analyses demonstrated the homogeneity of each dosage form analysed before the study (concentration range: 3 to 200 mg/mL). Furthermore, the nominal concentrations of the test material in the vehicle corresponded to the measured concentrations.

STABILITY
Each dosage form prepared in week 1 was stored at 4 °C (protected from light) and sampled after 0, 4 and 9 days storage. The aliquot sampled on day 4 was stored frozen at -20 °C pending analysis on the last sampling occasion (day 9) when all samples were analysed. In each case, the mean result of the homogeneity analysis (day 0) was taken as the initial value for the stability test.
The results of the analyses demonstrated a satisfactory stability of the same dosage forms over a 9-day period at 4 °C (protected from light).

ASSAY METHOD
An aliquot of each dosage form was diluted and analysed by High Performance Liquid Chromatography with Ultra-Violet detection at 205 nm. The concentration of the test material was determined from a calibration curve of peak area against concentration of test material in standard solutions (external standard calibration).

- Sample preparation
An aliquot (1 mL) of each dosage form under magnetic stirring was sampled (weighed accurately) in a tube (for group 1) and diluted five-fold in acetone/water (50/50, v/v) or in a 100 mL volumetric flask (for other groups) which was made to volume with acetone/water (50/50, v/v). Subsequently, the following dilutions were carried out in mobile phase to achieve target concentrations in the range of 1.5 to 6 μg/mL test material:
(i) 1:2 in mobile phase for group 1 (0 mg/mL)
(ii) 1:5 in mobile phase for group 2 (3 mg/mL)
(iii) 1:200 in mobile phase for group 3 (30 mg/mL)
(iv) 1:400 in mobile phase for group 4(200 mg/mL)
From the aliquot of dosage form sampled (weighed accurately), the real volume of the aliquot analysed was determined (taking into account the density of each preparation) and the value of the first dilution factor was calculated.

CHROMATOGRAPHIC CONDITIONS (HPLC/UV)
Pump: HP Series 1100 (Hewlett Packard) or 600E (Waters) or 114 M (Beckman)
Mobile phase: Acetonitrile 35 %, Water 65 %
Flow rate: 1 mL/min
Column: Zorbax RX C18 (Interchirn), particle size = 5 μm, length = 250 mm, inner diameter = 4.6 mm
Temperature: Room temperature
Detector: UV 486 or Dual λ Absorbance detector 2487 (Waters) or SpectraSeries UV 150 (TSP) SpetraMonitor 3100 (Milton Roy), wavelength = 205 nm
Injector: Autosamplers 717 or 717plus or Wisp 710B (Waters)
Injected volume: 20 μL
Data acquisition software: Multichrom 2 (Fisons Instruments)
Retention time of test material: Approximately 7.5 minutes
Analysis time: 10 minutes

- Calibration curve
Peak areas were determined for standard solutions ranging from 0.05 to 10 µg/mL of test material (six levels). A calibration curve was obtained by linear regression analysis of peak areas against concentrations. The regression analysis of the calibration data gave an equation of the following form:
Y = aX
Where:
Y = peak area of test material (μVs)
X = concentration of test material (mg/mL)
a = slope value

- Assay
Diluted samples of dosage form were analysed by HPLC. Each sample was diluted appropriately, and one injection was performed for each final dilution. The test material peak area was determined for each sample and the concentration of the test material calculated using the equation obtained from the calibration data.

- Results
Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the dosage forms administered since the deviations from nominal concentration were in the requested range of ± 10 %.

VALIDATION OF THE ANALYTICAL METHOD
The specificity, limit of quantification, linearity, repeatability of injections, accuracy and precision (Coefficient of variation: CV%) of the analytical method were determined.

- Specificity
The specificity of the analytical method was demonstrated by analysis of a standard solution of the test material in mobile phase and analysis of a solution of 1 % methylcellulose (vehicle) diluted ten-fold. No relevant interference between the test material peak and vehicle was observed on chromatograms.

- Limit of quantification
The limit of quantification of the analytical method was established as 0.05 μg/mL for a standard solution of test material. This limit corresponds to a limit of quantification of 0.0005 mg/mL for the test material in 1 % methylcellulose.

- Linearity
Linearity was checked by analysis of three different sets of seven standard solutions 0.05, 0.1, 0.5, 1, 2, 5, and 10 μg/mL of test material in mobile phase. Satisfactory linearity was obtained in the range of 0.05 to 10 μg/mL since the coefficients of determination obtained were higher than 0.999.

- Repeatability of injections
Replicate analysis (n = 10) of a solution containing 0.993 μg/mL of the test material gave satisfactory results since the coefficients of variation obtained were 1.7 % (based on peak height) and 2.1 % (based on peak area). Based on these results, the repeatability of injections of the analytical method was validated taking into account peak area.

- Accuracy and precision
Six analyses of a dosage form containing 3 and 200 mg/mL of the test material in water were carried out. An aliquot (1 mL) of each dosage form was sampled (weighed accurately) in a volumetric flask (100 mL) and the flask was made to volume with acetone/water (50/50, v/v). Samples were diluted with mobile phase before analysis.
The accuracy obtained was 92 and 99 % for the 3 and 200 mg/mL dose levels, respectively, and the precision (CV%) was 5 and 2 % for the 3 and 200 mg/mL dose levels, respectively.
The analytical method was validated and considered to be suitable for dosage forms analysis.

Duration of treatment / exposure:

For the males, dosing occurred for 10 weeks before mating and during the mating and post-mating periods until sacrifice.
For the females, dosing occurred 2 weeks before mating, during the mating period and during pregnancy and lactation until day 21 post-partum inclusive (or until sacrifice, for unmated females).

Frequency of treatment:

Once a day, seven days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 24 females females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were determined following the results of a previously conducted 4-week study conducted at 15, 150 and 1000 mg/kg/day. In this study, 150 mg/kg/day was a No Observable Effect Level and 1000 mg/kg/day was a No Observable Adverse Effect Level. Consequently, the same dose levels were selected for this study.
- Rationale for animal assignment (if not random): Before the beginning of the treatment period, the animals were allocated to the groups, according to a stratification procedure, so that the average body weight of each group was similar.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked at least twice a day, including during weekends and public holidays, for mortality and signs of morbidity. Any animal found dead or killed prematurely was subjected to a macroscopic examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed for clinical signs, including evidence of abortion, at least once a day, at approximately the same time (including evidence of abortion/resorption for the females).

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1, 7, 14 and 21 post-partum.

FOOD CONSUMPTION: Yes
The quantity of food consumed by each animal was recorded once a week, over a 7 or 8-day period, from the first day of treatment (except during the mating period) until day 14 postpartum. Food consumption was not recorded over days 14-21 post-partum, since the animals were fasted prior to necropsy. During the mating period, the food consumption was noted for neither males nor females. Food intake per animal and per day was calculated

WATER CONSUMPTION: No

OTHER: PARTURITION
Females were allowed to litter normally and rear their progeny until day 21 post-partum. Any sign of a difficult or prolonged parturition was recorded. The day of completed parturition was designated day 1 post-partum. The length of gestation was calculated.

Oestrous cyclicity (parental animals):

During mating, confirmation of mating was made in the morning by checking the presence of a vaginal plug or of sperm in a vaginal lavage. The stage of oestrous cycle was determined at that time, until mating was obtained.

Sperm parameters (parental animals):

Parameters examined in P males: The following organs were subjected to histopathological examination: coagulating gland, epididymides, prostate, seminal vesicles and testes.

Litter observations:

PARAMETERS EXAMINED
- Litter size: The total litter size and numbers of pups of each sex was recorded as soon as possible after birth. The litters were observed daily in order to note the number of live, dead and cannibalised pups. Any gross malformation in pups was noted.
- Body weight: The weight of each litter was recorded on days 1, 4, 7, 14 and 21 post-partum (individual weights were recorded on day 21 post-partum).
- Clinical signs: The litters were observed daily for clinical signs.

GROSS EXAMINATION OF DEAD PUPS
Dead pups, pups sacrificed and pups killed on day 21 post-partum were examined externally and internally for gross abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours of fasting, all surviving animals were asphyxiated by carbon dioxide and killed by exsanguination. The males were killed after the end of the mating period; the females and their pups were killed on day 21 post-partum. The females showing no evidence of mating were killed at least one week after the last day of the mating period.

GROSS NECROPSY
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. In the parent females, the corpora lutea and implantation sites were recorded. In the females which did not mate or were apparently non-pregnant, the presence of implantation scars on the uterus was checked using the Salewski staining technique.

PRESERVATION OF TISSUES
In all parent males and females, the macroscopic lesions and following tissues were preserved in 10 % buffered formalin ( except for the eyes and Harderian glands which were fixed in Davidson's fixative, and the testes and epididymides which were preserved in Bouin's fluid): Coagulating gland, epididymides, ovaries, pituitary gland, prostate, seminal vesicles, testes, uterus (horns and cervix) and vagina.

HISTOPATHOLOGY
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with haematoxylin-eosin. A microscopic examination was performed on the tissues listed above in all males and females suspected of being infertile (males or females which did not mate and mated non-pregnant females and the corresponding male).

Postmortem examinations (offspring):

SACRIFICE
On day 21 post-partum all surviving animals were asphyxiated by carbon dioxide in excess.

GROSS NECROPSY
Dead pups, pups sacrificed and pups killed on day 21 post-partum were examined externally and internally for gross abnormalities. There was no preservation of tissues.

Statistics:

Mean values were compared by one-way analysis of variances and Dunnett test (quantitative values) or by Fisher exact probability test (proportions). The values are considered as normally distributed and variances are considered as homogeneous.

Reproductive indices:

The mating index and fertility index were calculated.

Offspring viability indices:

The live birth index and viability index were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the treated groups, there were no clinical signs which were considered to represent adverse effects of the test material. For both males and females, ptyalism was attributed to treatment with the test material but was not considered to represent an adverse effect.

MALES
Loud breathing was recorded in two males of the 1000 mg/kg/day group; since this was observed to a small extent and only transiently (4 to 9 days), a relationship to treatment with the test material was ruled out. In the 150 and 1000 mg/kg/day groups, 7/12 males and 12/12 males, respectively, presented with ptyalism, generally after 11 to 12 days of treatment.

FEMALES
Signs of poor clinical condition (emaciation, piloerection, loud breathing, dyspnea, eyes half closed and/or round back) were noted in 1/24 control females and 4/24 females of the 1000 mg/kg/day group. In view of the slightly higher incidence in the high-dose group, a relationship to treatment with the test material cannot be excluded. Nevertheless, these findings observed transiently (principally during the lactation period) were considered to be of minimal toxicological significance. This conclusion was also supported by the absence of other signs of toxicity (e.g. no effect on food consumption or body weight gain). The mass recorded at the level of mammary glands in 1/24 control females, 1/24 females given 150 mg/kg/day and 1/24 females given 1000 mg/kg/day may correspond to galactocele. In the 1000 mg/kg/day groups, 11/24 females presented with ptyalism, starting at different times during the treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

MALES
There were no deaths in the control group or 15 mg/kg/day group. In the 150 mg/kg/day group, one accidental death was recorded, bearing no relationship to treatment with the test material. In the 1000 mg/kg/day group, one male was found dead on day 57 of treatment, having presented no noteworthy clinical signs prior to death (except for ptyalism) and no abnormality in food consumption or body weight gain. At necropsy, there were no macroscopic post-mortem changes. The cause of death of this animal was not established. Since this was the only death in the study, and in view of the absence of overt signs of toxicity, even at this dose-level, a relationship to treatment with the test material was considered to be improbable.

FEMALES
There were no deaths in any control or treated group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
The body weight gain of the treated males was similar to that of the control animals. The differences occasionally observed were slight, not statistically significant and/or not dose related; consequently, they were considered to be of no toxicological significance.

FEMALES
When compared to the control animals, the body weight gain of the treated females was similar during the pre-mating, mating and pregnancy periods but was lower during the beginning of the lactation period (Table 1).
A lower body weight gain was recorded during the first week post-partum only, a period when large variations of body weight are commonly recorded. In addition, the body weight gain in the treated groups was greater during the second and the third weeks of the lactation periods (this might be explained by a lower number of pups). Consequently, a relationship to treatment with the test material was considered to be improbable. This conclusion was also supported by body weights prior to terminal sacrifice which were similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MALES
The food consumption of all the treated males was similar to that of the control males, during the pre-mating and post-mating periods.

FEMALES
The food consumption of all the treated females was similar to that of the control females, during both pre-mating and pregnancy periods. During the lactation period, the food consumption was slightly lower in the treated groups (Table 2).
A lower food consumption was recorded during the first week post-partum (moderate, all groups), and the second week post-partum (slight, high-dose group only). Lactation is a period where large variations of this parameter are commonly recorded. In addition, the differences from the control group were not dose-related during the first week. Consequently, a relationship to treatment with the test material was considered to be improbable.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The notable findings observed in the sexual organs of the animals which were suspected to be infertile were as follows:
In the males of the 15 mg/kg/day group, minimal hyposecretion in the seminal vesicles and interstitial mononuclear cell aggregation in the prostate were seen in one animal while in the group receiving 1000 mg/kg/day one male showed moderate hyposecretion in the prostate and another minimal interstitial mononuclear cell aggregation in the prostate. No microscopic findings were noted for the other males examined.
Among the females, all three receiving 15 mg/kg/day and the two animals receiving 1000 mg/kg/day, showed findings consistent with pseudopregnancy. This was characterised by few corpora lutea in the ovaries, endometrial epithelial cell atrophy and compact endometrial stroma in the uterus and mucification of the vaginal epithelium. The remaining female, receiving 150 mg/kg/day, showed findings consistent with oestrus in the ovaries, uterus and vagina.
All the above findings were those which are commonly seen in the untreated laboratory rat of this strain and age and were considered to be of no toxicological importance.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The oestrous cycle stage was not affected at any dose-level, for either control or treated groups. There was only one female in the 1000 mg/kg/day group which did not mate but showed a pseudo-pregnancy. This common finding was not considered to be treatment-related.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

MATING INDEX
The mating index (mated/paired) was 100 % in all male groups and was 88 to 100 % in all female groups (a single female of the high dose group did not mate, but this was considered to be fortuitous). This conclusion was also supported by the absence of any treatment-related changes at microscopic examination of the genital organs of these animals.

PRE-COITAL INTERVAL
The pre-coital interval was similar in the control group and the treated groups. Most paired animals mated within 1 to 4 days of cohabitation, i.e. within the duration of a single oestrous cycle, except for one to three pairs per group, for which the duration was longer. This finding, commonly recorded at this low incidence, was not attributed to treatment with the test material.

FERTILITY INDEX
The male and female fertility index (pregnant/mated) was similar in the control and treated groups, ranging from 88 to 100 %, without indication of dose- or treatment-relationship. This conclusion was also supported by the absence of any treatment-related changes at microscopic examination of the genital organs of these animals.

DURATION OF GESTATION
The duration of gestation was similar in the control and the treated groups, and close to the normal value of 21 to 22 days.

DELIVERY DATA
The data collected at delivery of the pregnant females are summarised in Table 3.
The gestation index (females with liveborn pups/pregnant females) was similar in the control and treated groups. The number of implantation sites was also similar in the control and the treated groups, suggesting that ovulation and implantation of the ovum were not disturbed by treatment with the test material.
On the contrary, the post-implantation loss was higher in all the treated groups, leading to a number of pups delivered moderately lower in all the treated groups (-17 to -19 %). It cannot be established in such a study if this post-implantation loss was due to intra-uterine loss (resorptions or foetal deaths) or to stillborn pups which might have been totally cannibalised after birth. The higher post-implantation loss was considered to be related to treatment with the test material.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

general parental toxicity

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The test material was clinically well tolerated. Mating, fertility and gestation endpoints were not affected in any sex. Nevertheless, there was a slight to moderate increase of the post-implantation loss at all dose-levels.

2

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive performance

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No external abnormality was observed in any pups when they were first observed (i.e. on day 1 post-partum). There were no notable clinical signs in the pups during the lactation period.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There were no still born pups in any group. The survival of the pups was slightly lower in the 150 and 1000 mg/kg/day groups, principally during the first week of the lactation period (Table 4).
The higher post-natal mortality recorded in the 150 and 1000 mg/kg/day groups was considered to be related to treatment with the test material.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the treated groups, the body weight of the pups at birth (day 1 post-partum) was similar to that of the control pups, but the body weight gain was subsequently slightly lower (Table 5).
A lower body weight gain was recorded during the first week post-partum only. Since higher mortality was recorded during the same period in the treated pups, a relationship to treatment with the test material was considered to be probable.
The body weight gain in the treated groups was greater during the second and the third weeks of the lactation period; this might be explained by a lower number of pups per female.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no notable post-mortem macroscopic changes in the decedent pups that could have contributed to morbidity/mortality. In the pups killed on day 21 post-partum, the few findings recorded were among those commonly observed in rats.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

SEX RATIO
The sex ratio was similar in the control and the treated groups, and close to a theoretical value of 50 %.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOEL

Remarks:

teratogenic effects

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No teratogenic effects were seen

2

Key result

Dose descriptor:

NOEL

Remarks:

pre-natal and early post-natal development

Generation:

F1

Effect level:

< 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: Mean body weight (BW) and body weight gain (BWG) during lactation

Dose-level (mg/kg/day)	0	15	150	1000
BWG day 1 to 7 pp (g)	29	12**	8***	10***
BWG day 7 to 14 pp (g)	7	36***	32***	30***
BWG day 14 to 21 pp (g)	-66	-56	-60	-53*
BWG day 1 to 21 pp (g)	-29	-8***	-20	-13**
BW on day 21 pp (g)	313	310	313	314

*p<0.05

**p<0.01

***p<0.001

pp: post-partum

Table 2: Mean food consumption (lactation period), gram/day

Dose-level (mg/kg/day)	0	15	150	1000
Days 1 to 7
Mean	50	36***	36***	36***
Difference from controls (%)	-	-28	-28	-28
Days 7 to 14
Mean	77	73	69	66*
Difference from controls (%)	-	-5	-10	-14

*p<0.05

***p<0.001

Table 3: Summary of delivery data

Dose-level (mg/kg/day)	0	15	150	1000
Pregnant females	24	21	23	22
Females with liveborn pups	24	21	23	2
Gestation index (%)	100	100	100	100
Implantation sites (per female)	15.9	15.1	14.7	15.8
Pups delivered (per female)	15.1	12.6*	12.3*	12.6*
Post-implantation loss (%)	5.0	17.0	16.3	20.3

*p<0.05

Table 4: Summary of pup survival during lactation period

Dose-level (mg/kg/day)	0	15	150	1000
Pups delivered	363	264	284	277
Post-natal deaths (including cannibalised pups)
Days 1 to 7 pp	9 (2.5 %)	15* (5.7 %)	30*** (10.6 %)	32*** (11.6 %)
Days 8 to 14 pp	2 (0.6 %)	3 (1.1 %)	2 (0.7 %)	7*(2.5 %)
Days 15 to 21 pp	7 (1.9 %)	0* (0.0 %)	1 (0.4 %)	0* (0.0 %)
Total days 1 to 21 pp	18 (5.0 %)	18 (6.8 %)	33 (11.6 %)	39 (14.1 %)

*p<0.05

**p<0.01

***p<0.001

pp: post-partum

Table 5: Mean body weight (BW) body weight (gain) of pups during lactation

Dose-level (mg/kg/day)	0	15	150	1000
BWG day 1 to 7 pp (g)	7.8	6.0	6.5	6.1
BWG day 7 to 14 pp (g)	12.6	14.2	12.6	12.5
BWG day 14 to 21 pp (g)	9.5	12.5	11.1	11.3
BW on day 21 pp (g)	36.4	38.9	36.8	36.2

pp: post-partum

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the No Adverse Effect Level for paternal and maternal toxicity is higher than 1000 mg/kg/day. The No Effect Level for pre-natal and early post-natal development was lower than 15 mg/kg/day. The No Effect Level for teratogenic effects is higher than 1000 mg/kg/day.

Executive summary:

The potential for the test material to cause reproductive toxicity was investigated in accordance with 87/302/EEC, 18th November 1987 (or Annex V, 92/69/EEC) under GLP conditions.

The study evaluated the potential of toxic effects of the test material on gonadal function, mating behaviour, conception, developmental of the conceptus during gestation, parturition development and development of the pups during lactation.

Three groups of 12 male and 24 female Sprague-Dawley rats received the test material by oral gavage, once a day, at 15, 150 or 1000 mg/kg/day throughout the premating period (10 weeks), mating period and until final sacrifice in the males and throughout the premating period (2 weeks), mating period, pregnancy and lactation until final sacrifice (day 21 post-partum) in the females. A group of 12 males and 24 females was given the vehicle alone (aqueous methylcellulose at 1 %) and acted as a negative control group.

Mortality and clinical signs were checked daily. Body weight and food consumption were recorded at designated intervals. A mating trial was carried out in all animals: males and females were paired for 3-week maximum period, until mating was obtained. Oestrous cycle stage was recorded daily during this period. The females were allowed to deliver and to rear their progeny until weaning (day 21 postpartum). During the lactation period, the pups were examined daily for survival, external abnormalities and clinical signs. The body weight and sex were recorded.

At final sacrifice of the parent males and females, a macroscopic examination was carried out and the genital organs, accessory sex organs and pituitary gland were sampled. A microscopic examination was performed in the animals suspected to be infertile. At final sacrifice of the pups, both external and internal examinations were performed to search for gross abnormalities.

No deaths or clinical signs of toxicity were noted in the treated groups. There was only a moderate to high incidence of ptyalism at 150 mg/kg/day (males) and 1000 mg/kg/day (males and females); this sign was not considered to represent an adverse effect. The body weight gain and food consumption of the treated animals did not present differences from the control values which were considered to be of toxicological significance.

The mating, gestation and fertility indexes were similar in the control and the treated groups. The pre-coital interval and the duration of gestation were not notably different in the control and the treated groups. The oestrous cycle stages were similar in the control and treated groups.

The difference between the number of uterine implantation sites and the number of delivered pups showed, in all the treated groups, a slight to moderate increase of the post-implantation loss: 17.0, 16.2 and 20.4 % in the 15, 150 and 1000 mg/kg/day groups, respectively, vs. 5.0 % in the control group. This effect was considered to be related to treatment with the test material. Nevertheless, ovulation and implantation were not affected at any dose-level. No external abnormalities were observed in the pups at birth. The survival of the pups during the lactation period was similar in the control and 15 mg/kg/day group and was slightly lower in the 150 and l 000 mg/kg/day groups, principally during the first week after delivery. This effect was considered to be related to treatment with the test material. There were no clinical signs in the pups of any group during the lactation period. The body weight gain of the pups of the treated groups was slightly lower than that of the control pups during the first week of the lactation period. This finding was considered to be related to treatment with the test material. The sex ratio was similar in all the groups.

There were no macroscopic changes at examination of the parent males or females or the pups which were attributable to treatment with the test material. There were no treatment-related microscopic findings in the organs of the animals suspected of being infertile.

The test material was clinically well tolerated. Mating, fertility and gestation endpoints were not affected in any sex. Nevertheless, there was a slight to moderate increase of the post-implantation loss at all dose-levels, a slight reduction of the survival of the pups at 150 and 1000 mg/kg/day and a slight reduction of growth at 15, 150 and 1000 mg/kg/day. No evidence of teratogenic effects was observed at any dose-level.

Under the conditions of this study, the No Adverse Effect Level for paternal and maternal toxicity is higher than 1000 mg/kg/day. The No Effect Level for pre-natal and early post-natal development was lower than 15 mg/kg/day. The No Effect Level for teratogenic effects is higher than 1000 mg/kg/day.