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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 422 study was performed with 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol. The No Observed Adverse Effect Level (NOAEL) for parental toxicity was 300 mg/kg/day as well as the NOAELs for reproductive performance and for toxic effects on progeny.

Link to relevant study records

Reference

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.

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

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

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

actual ingested

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)

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)

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: 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.

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

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

Critical effects observed:

no

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Critical effects observed:

no

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good, NOAEL derives from an OECD 422.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

One OECD 422 was available with 2,2'-Isopropylidenebis(p-phenyleneoxy)diethanolt hrough 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 5p.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 20p.c.) periods and during lactationon Days 1 and 5p.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 5p.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 5p.p..

The males were sacrificed after at least 5 weeks of treatment and the females on Day 6p.p.. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopicpost-mortemexamination 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 macroscopicpost-mortemexamination of the principal thoracic and abdominal organs was performed on all pups sacrificed on Day 5p.p..No tissues were preserved.

Results

 

The test item concentrations in dose formulations analyzed were within an acceptable range of variationswhen 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 6p.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 4p.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 5p.p.(6.7 g and 10.4 gvs.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 gvs.+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 gvs.+46, p<0.05).

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

 

There were notest item treatment-relatedeffects on mating, fertility and gestation indexes, on mean pre-coital time, on pup clinical condition andon pup sex ratio.There were notoxicologically significanteffects 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.

Effects on developmental toxicity

Description of key information

A prenatal developmental study via the oral route in rats is currently ongoing. The final report is due to be available in January 2023.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 30 March 2022
Final report date: 4 January 2023 (see attached justification)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

Supplier Females: Charles River (Italy).

Males: Stock animals obtained from Charles River (Italy). Males were unrelated to females.

Number of animals 98 females (88 on study and 10 spares).

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 33 days before commencement of pairing.

Age of the animals at the start of the study (Day 0 of gestation) 83 to 89 days of age.

Weight range of the animals at the start of the study (Day 0 of gestation) 260 to 333 g.

Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used were used during the acclimatization and gestation periods.

Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization Up to four females.
During pairing One (stock) male and one female.
Gestation One female.

Environmental Enrichment
Aspen chew block A soft white untreated wood block: provided to each cage throughout the study and replaced when necessary.

Diamond twists (paper product) Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding, Aspen chew blocks and Diamond twists.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

Method of preparation
Formulations were prepared under yellow light.

Suspension of the test item was performed following the steps below:

• Weighed the required quantity of test item.
• Ground the test item using a mortar and pestle.
• Added vehicle dropwise and mixed with the test item to obtain a homogeneous mixture.
• Progressively added the vehicle and transferred the mixture into a gauged flask, with rinsing of the mortar and pestle using vehicle.
• Homogenized the suspension by manual stirring before completing to final volume with vehicle.
• Homogenized the suspension by magnetic stirring for at least 10 minutes before further homogenisation using ultraturrax at 11000 rpm for 10 minutes.
• Further stirred the formulations by magnetic stirring at room temperature and protected from light for at least 15 minutes.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation Groups 1 to 3: On two occasions and dose formulations were prepared six days in advance of dosing.

Group 4: Daily on each morning of administration.

Storage of dose formulation Groups 1 to 3: Refrigerated (2 to 8°C) until required for use.

Group 4: Ambient (15 to 25°C).

Test item accounting
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Labcorp Study No. 8468033 In that study, formulations in the range 2 to 200 mg/mL were determined to be stable for:

• One day at ambient temperature (15 to 25°C)
• 15 days when stored refrigerated (2 to 8°C)

Achieved concentration Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.

Details on mating procedure:

Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smear.

Day 0 of gestation When positive evidence of mating was detected.

A colony of stock males from the same strain and source (CD IGS) was maintained specifically for the purpose of mating. These animals were not part of the study and were maintained as stock animals.

Allocation and Identification
Allocation On day of positive evidence of mating. Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Duration of treatment / exposure:

Females Day 6 to 20 after mating

Frequency of treatment:

Females were treated from Day 6 to Day 20 (inclusive) after mating, once daily at approximately the same time each day

Duration of test:

Study initiation (Study Plan signed by Study Director) 24 March 2022
Experimental start date (Animal arrival) 30 March 2022
Pairing commenced 02 May 2022
Treatment commenced 09 May 2022
Necropsy 24 to 27 May 2022

Dose / conc.:

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

Remarks:

Group 1 - Control. Vehicle only

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

22 females per dose group, including control

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Selection of the doses was based on the results of the preliminary embryo-fetal study (Labcorp study no. 8468035). During this study, the test item was administrated at doses of 500, 750 or 1000 mg/kg bw/day.

No mortality was recorded at all dose-levels. There were no clear treatment-related changes in clinical condition at any dose levels investigated, with only non-adverse and transient signs observed at 750 or 1000 mg/kg bw/day.

Following the commencement of treatment on Day 6 of gestation, when compared to Controls, a group mean body weight loss was observed following the first dose administration in pregnant females at all dose levels investigated, generally followed by slightly low body weight gains throughout the remainder of treatment at 750 or 1000 mg/kg bw/day. An overall low mean body weight gain (GD 6-21) for all groups of treated females (7%, 13% or 24% lower than Controls at 500, 750 or 1000 mg/kg bw/day, respectively) was recorded.

In accordance with the OECD Testing Guideline 414, the highest dose level was chosen with the aim to induce some developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering.

Based on the outcome of the preliminary study, the highest dose level was 1000 mg/kg bw/day, as it was the limit dose for the OECD TG 414, and it did not induce death nor severe suffering. 250 and 500 mg/kg bw/day were selected as the low and intermediate dose-levels, respectively.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:

Occasion Animals
At termination All adults surviving to scheduled termination on GD 21

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)

Sequence of blood sampling on each occasion
To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.

Blood sample site Sublingual vein.

Anesthetic Isoflurane.

Anticoagulant None.

Blood tubes Greiner Minicollect tubes with clotting activator.

Blood volume 1.0 mL.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for ten minutes at 4°C.

Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes and then mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided into two aliquots.

Number of aliquots
Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH

Final storage conditions Deep frozen (approximately -60°C to -90°C) pending analysis.

Fate of samples Aliquot 1 (T3 and T4): dispatched to the Department of LC MS/MS Bioanalysis, Labcorp.

Aliquot 2 (TSH): dispatched to the Department of Immunology and Immunotoxicology, Labcorp.

T3 and T4 Performed by the Department of LC-MS/MS Bioanalysis, Labcorp.

TSH Performed by the Department of Immunology and Immunotoxicology, Labcorp.

:

Maternal examinations:

Serial Observations

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

A complete necropsy was performed in all cases.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

• Pre-dose observation
• One to two hours after completion of dosing
• As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal, weekly during acclimatization and on Days 0, 3, 5, 12, 18 and 21 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization and on Days 0, 3 and 6 to 21 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 21 after mating.

Sequence To allow satisfactory inter-group comparison.

Tissue and regions examined
Abnormalities
Gravid uterine weight (including cervix and ovaries)
Thyroid

Organ Weights
For bilateral organs, left and right organs were weighed together.

Light Microscopy
Tissues preserved for examination were examined as follows:

Category Animals Tissues
Premature deaths All animals from all groups. Thyroid
Scheduled kill All animals from all groups. Thyroid

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

For each ovary/uterine horn
Number of:
Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

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

Apparently non pregnant animals and for apparently empty uterine horns The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, 1964)].

Blood sampling:

Blood samples were collected at the following occasion:
Occasion Animals
At termination All adults surviving to scheduled termination on GD 21

Blood volume 1.0 mL.

Fetal examinations:

Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

50% of fetuses in each litter Sexed internally, examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.

50% of fetuses in each litter Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).

Processing Heads were removed from Bouin’s fixed fetuses and were subject to Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution.

IMS fixed fetuses were processed and stained with Alizarin Red and Alcian Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red and Alcian Blue stained fetuses Assessed for skeletal development, cartilage and abnormalities.

Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

Ano-Genital Distance
Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:

Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.

Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.

Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.
In the fetal examination appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistics:

Please refer to "any other information on materials and methods"

Indices:

Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) / Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses) / Number of implantations)) x 100

All group values and SD were calculated from the individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Administration of 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol to the surviving females given 1000 mg/kg/day or in females given 250 or 500 mg/kg/day was generally well-tolerated with no clear treatment-related changes in clinical condition observed. A slight increased incidence of piloerection immediately following dosing was generally observed from Gestation Day 10 in females given 1000 mg/kg/day and from Gestation Day 18 in females given 500 mg/kg/day although this was not associated with further clinical signs, transient and not considered adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two premature deaths occurred during the study:
Female 4F 75 given 1000 mg/kg/day was euthanized for welfare reasons on Gestation Day 8 following post-dose observations on Gestation Day 7 that consisted of piloerection, hunched posture and decreased activity and a 20 grams body weight loss. Macroscopic examination revealed firm abnormal contents in the cecum and rectum with dark areas in the stomach glandular mucosa and this female was pregnant with 11 live fetuses.
Female 4F 82 given 1000 mg/kg/day was euthanized for welfare reasons on Gestation Day 12 due to poor clinical condition with post-dose observations previously seen were piloerection and unsteady gait with an overall body weight loss of 51 grams and was further associated with low food consumption. Macroscopic examination did not reveal any abnormalities although this female was found with 15 early resorptions (total litter resorption).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment on Day 6 of gestation, when compared to Controls, a group mean body weight loss was observed following the first dose administration in pregnant females given 1000 mg/kg/day and was followed by periods of slightly low body weight gains during the remainder of treatment. These differences resulted in an overall low group mean body weight gain (GD 6-21) for females given 1000 mg/kg/day attaining statistical significance (9% lower than Controls). Females receiving 500 mg/kg/day showed a statistically significantly lower overall body weight gain when compared to Controls on GD 6-7 (p<0.05). There was no effect of treatment on body weight or body weight gain in females given 250 mg/kg/day.

Mean gravid uterine weight on Day 21 of gestation was not affected by treatment at any dose level investigated. When overall group mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight gain was low at 1000 mg/kg/day attaining statistical significance when compared to Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment, when compared to Controls, females given 1000 mg/kg/day showed a statistically significantly low group mean food consumption during Day 6-9 of gestation with food intake during the remainder of treatment was similar to Control values although these differences resulted in an overall low group mean food consumption at this dose level. Food consumption in females given 250 or 500 mg/kg/day was not affected by treatment.

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:

no effects observed

Description (incidence and severity):

The assessment of thyroid gland organ weight parameters at scheduled termination did not reveal any differences that were considered related to treatment at any dose level investigated.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination at scheduled termination did not reveal any treatment-related findings in maternal females at all dose levels investigated.

All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague-Dawley rats of this age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic findings in the thyroid gland.
All microscopic findings were considered spontaneous and/or incidental because they
occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague-Dawley rats of this age.

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

Not applicable as rats do not abort

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on litter data, as assessed by the mean number of implantations, resorptions (early or late), live young, sex ratio or the levels of pre- and post-implantation loss at all dose levels investigated.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Remarks on result:

other: Not yet confirmed as report in draft stage

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment of mean placental weights, total litter weights, or male, female and overall fetal weights at any dose level investigated.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on live young

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no evidence of an effect of maternal treatment on sex ratio

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There was no effect of treatment of fetal ano-genital distance.

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major abnormalities, minor skeletal abnormalities and skeletal variants showed no relationship to treatment.
Across the treated groups, there was an increased fetal incidence of variant of short supernumerary 14th ribs, when compared to concurrent Control, and was outside the fetal Historical Control Data (HCD) range at 1000 m/kg/day (30 fetuses in 10 litters) but was within litter HCD range; this variant was not considered an adverse finding.
At 1000 mg/kg/day, there was an increase in fetal incidence of variant of incompletely ossified 5th and/or 6th sternebrae, when compared to concurrent Control, but was within fetal and litter HCD range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and not considered adverse.

Visceral malformations:

no effects observed

Remarks on result:

other: Not yet confirmed as report in draft stage

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The conclusion will be added upon completion of the examinations currently conducted on the test item.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available (further information necessary)

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

The conclusion will be added upon completion of the examinations currently conducted on the test item as part of the study conducted in accordance with the OECD Testing Guideline 414.

Justification for classification or non-classification

Based on the available information from the reproscreening study, 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol does not have to be classified as toxic for reproduction in accordance with the criteria outlined in Annex I of 1272/2002/EEC.

 

Conclusion on classification will be amended upon receiving the final report on the OECD 414.