2,2'-isopropylidenebis(p-phenyleneoxy)diethanol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2015 -- 01 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

2,2'-isopropylidenebis(p-phenyleneoxy)diethanol

EC Number:

212-985-6

EC Name:

2,2'-isopropylidenebis(p-phenyleneoxy)diethanol

Cas Number:

901-44-0

Molecular formula:

C19H24O4

IUPAC Name:

2,2'-isopropylidenebis(p-phenyleneoxy)diethanol

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: breeder: Charles River Laboratories Italia, Calco, Italy.- Age/Weight: at the beginning of the treatment, the males were 10 weeks old and had a mean body weight of 435 g (range: 382 g to 483 g) and the females were 9 weeks old had a mean body weight of 237 g (range: 193 g to 272 g). - Housing: polycarbonate cages- Diet: SSNIFF R/M-H pelleted diet (free access)- Water: tap water filtered with a 0.22 µm filter (free access)- Acclimation period: for a period of 7 days before the beginning of the treatment period. ENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2°C- Humidity (%): 50 ± 20%- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air- Photoperiod (hrs dark / hrs light): 12 h/12 h.IN-LIFE DATES: 13 October 2015 to 01 December 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) methylcellulose 4000 cps aqueous solution

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:Type of formulation (visual observation): solution in the vehiclePreparation:- weigh the required quantity of test item,- ground the test item using a mortar and pestle,- add a few drops of vehicle and mix with the test item to obtain a homogeneous mixture,- progressively add the vehicle and transfer the mixture into a gauged flask,- rinse the mortar and pestle and add the rinsing liquid into the gauged flask,- homogenize the suspension by manual stirring before completing to final volume with vehicle,- homogenize the suspension by magnetic stirring for at least 10 minutes,- homogenize the suspension by ultraturrax at 11000 rpm for 10 minutes,- stir the formulations by magnetic stirring at room temperature and protected from light for at least 15 minutesVEHICLE- Justification for use and choice of vehicle: suitable formulation in the vehicle- Concentration in vehicle: 20, 60 and 200 mg/mL- Amount of vehicle (if gavage): 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1- Length of cohabitation (mating period): until mating occurred- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 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:

Type of method: HPLC/UVTest item concentrations: remained within an acceptable range of variation compared to nominal values when analysed in Weeks 1, 3 and 6

Duration of treatment / exposure:

In the males:- 2 weeks before mating,- during the mating period,- until sacrifice (5 weeks of treatment in total),In the females:- 2 weeks before mating,- during the mating period,- during gestation,- during lactation until Day 5 p.p. inclusive for lactating females,- or until sacrifice for females with no delivery by Day 26 p.c..

Frequency of treatment:

Daily

Details on study schedule:

- No F1 parents (only one generation mated)- Age at mating of the mated animals in the study: 11-12 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:The dose-levels were selected in agreement with the Sponsor, and are based on the results of a previous study. In this study, five male and five female Sprague-Dawley rats were given the test item at 100, 300 or 1000 mg/kg/day for 2 weeks.There were no unscheduled deaths and the only test item treatment-related clinical sign was ptyalism in all animals at 1000 mg/kg/day. There were no toxicologically relevant test item-related changes in mean body weight. When compared with controls, mean body weight gains were transiently lower in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day during the first week of treatment which was considered as non-adverse. Minor toxicologically significantly lower mean food consumption was observed in males at 1000 mg/kg/day in the first week of treatment. The test item administration induced minimal to slight increases in mean liver weights at 1000 mg/kg/day but there were no macroscopic changes related to the test item administration at any doses.Therefore, the same dose-levels were used in the present study.- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required)

Examinations

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:- Time schedule: at least twice a day during the treatment period.CLINICAL OBSERVATIONS:- Time schedule: once a day during the treatment period.DETAILED CLINICAL SIGNS- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study. BODY WEIGHT:- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.FOOD CONSUMPTION:- Time schedule: Males: on the first day of treatment, then once a week until the start of the mating period. Females: on the first day of treatment, then once a week until mating, on Days 0, 7, 14 and 20 post-coitum and Days 1 and 5 post-partum.REPRODUCTION (apart from indices):- Pre-coital time and duration of gestation were recorded.

Oestrous cyclicity (parental animals):

fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:- weighing and microscopic examination: see Tissue Procedure Table below- a detailed examination of the testes was performed for control and high-dose males (groups 1 and 4), using a thorough understanding of tubule development through the different stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS: NoPARAMETERS EXAMINED:- number and sex of pups,- number of live, dead and cannibalized pups,- presence of gross anomalies, weight gain, clinical signs

Postmortem examinations (parental animals):

SACRIFICE- Male animals: all surviving animals after 5 weeks of treatment- Female animals: all surviving animals = on Day 6 post-partum (p.p.) or, for females which had not delivered, on Day 26 p.c. (after a body weight recording to check for a possible un noticed delivery).ORGAN WEIGHTSsee table below.GROSS PATHOLOGYA complete macroscopic post-mortem examination was performed on all animals.HISTOPATHOLOGY- all tissues listed in the Tissue Procedure Table from the first five sacrificed as scheduled males and the first five females sacrificed on Day 6 p.p. from the control and high-dose groups (groups 1 and 4),- thymus (females), spleen (males), liver (both sexes), kidneys (females) and adrenals (females) from the first five males sacrificed as scheduled and the first five females sacrificed on Day 6 p.p., from the low- and mid-dose groups (groups 2 and 3),- all macroscopic lesions from all groups,- all tissues listed in the tissue Procedure Table from all animals that died or were sacrificed prematurely,- reproductive organs from the four animals that did not conceive, to investigate possible causes.

Postmortem examinations (offspring):

SACRIFICE: on Day 5 post-partumGROSS NECROPSY: on all pups (surviving, prematurely sacrificed and found dead)HISTOPATHOLOGY: NoORGAN WEIGHTS: No

Statistics:

Statistical analyses were performed on body weight, food consumption, reproductive, hematology, blood biochemistry and organ weight data.

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora luteaPost-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantationsMating index = 100 * (Number of mated animals / Number of paired animals)Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)Viability index on Day 4 p.p. = 100 * (Number of surviving pups on Day 4 p.p. / Number of live born pups)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see Table 1Ptyalism was noted at 300 and 1000 mg/kg/day in a dose-related manner (incidence and duration) and was ascribed to the test item treatment.At 1000 mg/kg/day, piloerection and round back were observed in both sexes in several animals. One female was pale and cold to the touch at the same time on the first days of lactation (low food consumption was concomitantly recorded in this female) and reminded clinical signs noted in the prematurely sacrificed female in lactation. All these clinical signs were considered to be test item treatment related.At 300 mg/kg/day, there was 1/20 animals with piloerection and round back in premating and gestation period, and one other female with piloerection on Days 5 and 6 p.p.. There were no test item treatment-related clinical signs at 100 mg/kg/day.The other clinical signs recorded in the study (chromorhinorrhea, cutaneous lesion, area of hair loss, soft feces, necrosed tail, scab, short tail, reflux at dosing) were not considered to be test item treatment-related: there were in limited incidences and/or are of common background of laboratory rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no premature deaths in males.At 1000 mg/kg/day:- one female was prematurely sacrificed on Day 6 due to poor clinical condition (piloerection, round back, emaciated appearance, dehydration, hypoactivity and dyspnea). Reduced size of thymus and spleen and distended colon/rectum were observed. Lymphoid atrophy was noted in the thymus (cortex and medulla), in the spleen (all compartments) and in lymph nodes (mesenteric and mandibular). Slight hepatocellular hypertrophy and slight tubular vacuolation, dilated tubules along with hyaline casts in the kidney were also recorded. The cause of death was not evident but the lesions in kidney may have been a contributing factor,- one other female was prematurely sacrificed during lactation on Day 2 p.p.. due to poor clinical condition (piloerection, emaciated appearance, pallor of extremities, cold to the touch, hypoactivity, bent head, locomotory difficulties and dyspnea; loss of 9% of body weight from Days 1 to 2 p.p.). 67% of its pups did not have milk in the stomach on the day of sacrifice. Brown contents were observed in the female stomach, which was associated with multifocal erosion of the mucosa on microscopic examination. Likewise female, lymphoid atrophy was observed in several immune tissues, including thymus (cortex and medulla), spleen (all compartments), and lymph nodes (mesenteric and mandibular). Minimal tubular vacuolation in the kidney were also recorded. The cause of death was not evident.Both deaths were likely related to the test item treatment (both at the high-dose).At 300 mg/kg/day: One female was prematurely sacrificed on Day 22 p.c. due to difficulties to deliver. The female had on that day piloerection, pallor of extremities and what was thought by the animal technician to be a fetus blocked in the vagina but which was in fact a vaginal septum transverse (seen at necropsy) preventing the delivery. Brown translucent contents were observed in both uterine horns, along with one implantation scar, three live and one dead fetuses in the right horn and two live and one dead fetuses in the left one. Vagina was enlarged and contained brown, translucent materials. As the vaginal septum transverse was a congenital anomaly, these difficulties to deliver and death were not considered to be test item treatment-related.There were no other premature deaths of females in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see Table 2At 1000 mg/kg/day and when compared with controls:- in males, statistically significantly lower mean body weight was noted as soon as in the first week of treatment (down to -10% vs. controls at termination). Mean body weight gain was statistically significantly lower in the first week of treatment and over the entire treatment period,- in females, mean body weight was statistically significantly lower during gestation and lactation (down to -12% vs. controls at the beginning of lactation). Mean body weight gain was particularly low in the first week of treatment as in males.These effects were not considered to be adverse as they were of slight amplitude.At 100 and 300 mg/kg/day, when compared with controls, mean body weight gains of males were statistically significantly lower in the first week of treatment and/or over the entire treatment period. These effects were considered to be of limited toxicological significance; mean body weights were not toxicologically affected. There were no toxicologically significant effects in mean body weight or mean body weight gain in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see Table 3At 1000 mg/kg/day and when compared with controls, mean food consumption was statistically significantly lower during the first week of treatment in both sexes. These variations were considered to be non-adverse as there were not severe and transient.There were no test item treatment-related effects on mean food consumption at 100 and 300 mg/kg/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see Part 7.5.1

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see Part 7.5.1

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see Table 5Prematurely dead ratsTwo females treated at 1000 mg/kg/day were euthanized prematurely on ethical grounds.Multifocal foci of mucosal erosion were noted in one female, which correlated well with brown contents on gross examination. Minimal tubular vacuolation in the kidney were also recorded. Lymphoid atrophy was observed in several immune tissues, including thymus (cortex and medulla), spleen (all compartments), and lymph nodes (mesenteric and mandibular). One likewise female, lymphoid atrophy was noted in the thymus (cortex and medulla), in the spleen (all compartments) and in lymph nodes (mesenteric and mandibular) of one female. Slight hepatocellular hypertrophy and slight tubular vacuolation, dilated tubules and hyaline casts in the kidney were also recorded. The findings in the liver (hypertrophy correlated with higher liver weights) and kidney (at least the vacuoles) were attributed to treatment with the test item. The lymphoid atrophy was considered to be possibly related to poor health condition and the associated stress.One female treated at 300 mg/kg/day was euthanized prematurely on ethical grounds. Periportal liver hypertrophy was observed, along with hematopoietic foci. Extramedullary hematopoiesis was marked in the spleen. These findings were also noted in the surviving rats and were considered to be test item treatment-related. Terminal sacrificeLiverMinimal to slight hepatocellular hypertrophy (centrilobular to diffuse) was observed in all males and females at 1000 mg/kg/day and in 1/5 male at 300 mg/kg/day. This microscopic finding was not considered to be adverse, but rather adaptive. Hematopoietic systemExtramedullary hematopoïesis was decreased in the spleen from males at 300 and 1000 mg/kg/day and there was an increase of hematopoietic foci in the liver from females treated at 1000 mg/kg/day, which correlated well with a trend toward decrease in reticulocyte counts in males and an increase in females. KidneyMinimal tubular vacuolation was observed in the kidneys from females from 100 mg/kg/day onwards, in a dose-related manner. ThymusThere was a trend toward lymphoid atrophy in the thymus (decreased cortex and medulla) in females treated at 1000 mg/kg/day. AdrenalsThere was a trend toward decreased cortical cell size in adrenals from females treated at 1000 mg/kg/day, which correlated with lower adrenal weights. This was better observed in the zona reticularis. Reproductive organsReproductive organs from two females and two males that did not conceive were examined. No abnormalities were detected.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

performed only for mating, not enough cycles examined

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

no in-vivo observation of sperm

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on mating, fertility and gestation indexes and on mean pre-coïtal time.There were no toxicologically relevant effects on delivery data.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item treatment-related clinical signs in pups (isolated findings and/or of common background).

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see Table 6At 1000 mg/kg/day, one litter was especially affected, with seven pups found dead on Day 1 or 2 p.p. and two pups found cannibalized on Day 2 p.p. (bad maternal care likely, the dam was not in good clinical condition on the first days of lactation).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see Table 7At 1000 mg/kg/day and when compared with controls, mean body weight of pups on Day 1 and 5 p.p. had a tendency to be lower. An effect of the test item treatment on mean pup body weight was not excluded.The lower mean body weight gain was mainly due to one litter (from one female which was not in good clinical condition on the first days of lactation).At 100 and 300 mg/kg/day, there were no effects on mean pup body weight and body weight gain at the beginning of the lactation period.

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, treatment-related

Description (incidence and severity):

see Table 8At 1000 mg/kg/day, there was one litter with three pups having a whitish thick fluid in the urinary bladder and one pup with an umbilical hernia. These necropsy findings occurred in one litter only but in the high dose group. A test item treatment effect cannot be totally excluded.At 100 and 300 mg/kg/day, there were no test item treatment-related necropsy findings in scheduled sacrificed pups.In prematurely dead pups, autolysis and absence of milk in the stomach observed at necropsy were of common background in prematurely dead rat pups in this kind of study. Short innominate artery and fluid filled abdomen can be seen spontaneously in Sprague-Dawley rats and were considered as incidental (isolated findings, no dose-relationship).

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratioThere were no effects on pup sex ratio in any groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Progeny toxicity

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 5 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day. Based on the experimental conditions and results of this study:- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the deaths and the accumulation of effects (clinical signs, effects on mean body weight, food consumption, hematology and blood biochemistry data, pathology findings) at 1000 mg/kg/day,- the NOAEL for reproductive performance was considered to be 1000 mg/kg/day, based on the absence of toxicologically significant effects on mating, fertility and delivery data up to this dose,- the NOAEL for toxic effects on progeny was considered to be 300 mg/kg/day, based on the effects on pup viability in a context of severe maternal toxicity and on pup body weight at 1000 mg/kg/day.

Executive summary:

The objective of this GLP study was to evaluate the potential toxic effects of the test item, following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 5 post-partum (p.p.).

This study provides initial information on possible effects:

.  on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus, and parturition.

 

Methods

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 p.p.. The test item was administered at dose-levels of 100, 300 and 1000 mg/kg/day in the vehicle [0.5% (w/v) methylcellulose 4000 cps aqueous solution]. Another group of ten males and ten females received the vehicle, alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

 

The concentration of the dose formulations was checked in study Weeks 1, 3 and 6.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating) and gestation (0, 7, 14 and 20 p.c.) periods and during lactationon Days 1 and 5 p.p..

The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p.. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on Days 1 and 5 p.p..

The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on several organs from five males and five females in the control and high-dose groups, on thymus, spleen, liver, kidneys and adrenals from five males and/or females in the low- and mid-dose groups, on all macroscopic lesions and reproductive organs from animals that did not conceive.

A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups sacrificed on Day 5 p.p.. No tissues were preserved.

 

Results

 

The test item concentrations in dose formulations analyzed were within an acceptable range of variations when compared with the nominal values (nominal concentration ± 15%). There was no test item observed in the control dose formulations.

 

At 1000 mg/kg/day, the test item treatment-related effects are described below:

.         there were two females prematurely sacrificed on Day 2 or 6 p.p. following poor clinical condition (piloerection, emaciated appearance, hypoactivity, dyspnea, etc) which were likely related to the test item treatment,

.         ptyalism was noted in all animals at several occasions; piloerection and round back were observed in both sexes, and one female was also pale and cold,

.         mean body weight was lower than in controls from the first week of treatment in males (down to -10%, p<0.01) and during gestation and lactation in females (down to -12%, p<0.01),

.         mean food consumption was lower than in controls during the first week of treatment in both sexes (-22 to -24%, p<0.01 or 0.001),

.         there were higher mean liver weight in both sexes and lower mean adrenals (females) and spleen (males) weights, as well as a tendency towards lower mean thymus weights in females,

.         minimal to slight hepatocellular hypertrophy was observed in both sexes, andminimal renal tubular vacuolation and a trend towards lymphoid atrophy in the thymus or towards decreased cortical cell size in adrenals in females. Extramedullary hematopoïesis was decreased in the spleen from males while increase of hematopoietic foci was noted in the females and correlated with reticulocyte count,

.         pup viability index was lower on Day 4 p.p. (86.1% vs. 98.7% in controls) because of a high rate of death in one litter following maternal toxicity,

.         mean body weight of pups on Day 1 and 5 p.p. (6.7 g and 10.4 g vs. 7.1 g and 11.4 g, respectively, both sexes together) had a tendency to be lower than controls,

.         one litter with three pups had a whitish thick bladder liquid and one pup with an umbilical hernia. A test item treatment effect cannot be excluded.

 

At 300 mg/kg/day, ptyalism was also noted in about half of the animals and two females had piloerection plus round back for one of them. Mean body weight gains of males were lower in the first week of treatment (+32 g vs. +46, p<0.05) and over the entire treatment period when compared with controls.

Increased absolute and/or relative-to-body mean liver weights were recorded. This finding correlated with hepatocellular hypertrophy only in one male. Lower absolute and/or relative-to-body mean spleen weights were observed in males, which was associated with a trend toward decreased hematopoïsesis in the red pulp.

 

Minimal tubular vacuolation was observed in the kidneys from 2/5 females.

 

At 100 mg/kg/day and when compared with controls, mean body weight gains of males were lower in the first week of treatment (+32 g vs. +46, p<0.05).

Minimal tubular vacuolation was observed in the kidneys from 1/5 females.

 

There were no test item treatment-related effects on mating, fertility and gestation indexes, on mean pre-coital time, on pup clinical condition andon pup sex ratio.There were notoxicologically significant effects on mean duration of gestation, mean pre- and post-implantation losses and mean number of pups delivered.

 

Conclusion

 

The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 5 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

 

Based on the experimental conditions and results of this study:

- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 300 mg/kg/day, based on the deaths and the accumulation of effects (clinical signs, effects on mean body weight, food consumption, hematology and blood biochemistry data, pathology findings) at 1000 mg/kg/day,

- the NOAEL for reproductive performance was considered to be 1000 mg/kg/day, based on the absence of toxicologically significant effects on mating, fertility and delivery data up to this dose,

- the NOAEL for toxic effects on progeny was considered to be 300 mg/kg/day, based on the effects on pup viability in a context of severe maternal toxicity and on pup body weight at 1000 mg/kg/day.