Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-[(1-methylethylidene)di-4,1-phenylene]bis[.omega.-hydroxy-, mixed acrylates and isononanoates

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2013 -- 03 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid

EC Number:

923-007-9

IUPAC Name:

4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid

Constituent 2

Reference substance name:

Esterification products of 4,4'-Isopropylidenediphenol, ethoxylated and prop-2-enoic acid and 3,5,5-trimethylhexanoic acid

EC Number:

919-846-5

Cas Number:

1187203-83-3

Molecular formula:

Not available for this UVCB.

IUPAC Name:

Esterification products of 4,4'-Isopropylidenediphenol, ethoxylated and prop-2-enoic acid and 3,5,5-trimethylhexanoic acid

Test material form:

other: colorless viscous liquid

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 at study initiation: approximately 10 weeks old for the males, and 9 weeks for the females
- Mean body weight at study initiation: 378 g (range: 333 g to 427 g) for the males, and 237 g (range: 216 g to 259 g) for the females
- Housing: in polycarbonate cages (Tecniplast 2154, 940 cm²)
- 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 8 days before the beginning of the treatment period. .

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 09 July 2013 to 01 September 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of vehicle and then homogenized using a magnetic stirrer at least 15 minutes in order to prepare an emulsion.
The test item dose formulations were prepared daily and were delivered at room temperature.

VEHICLE
- Justification for use and choice of vehicle: The vehicle used was sesame oil, batch Nos. MKBF2900V, MKBN1104V, MKBH4400V and MKBL4928V.
- Concentration in vehicle: 0, 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-UV
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: the homogeneity was determined on a range of dose formulations prepared at levels which cover the lowest and highest concentrations proposed for use in this study.
Stability: dose formulations were prepared daily

Duration of treatment / exposure:

In the males:
- 2 weeks before pairing,
- during the pairing period (up to 3 weeks),
- until sacrifice (at least 5 weeks in total),

In the females:
- 2 weeks before pairing,
- during the pairing period (up to 3 weeks),
- during gestation,
- during lactation until day 5 p.p. inclusive,
- until sacrifice for females with no delivery.

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

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
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, following the results of a previous 2-week toxicity study at dose-levels of 100, 300 and 1000 mg/kg/day.
In this study, ptyalism (not considered as adverse) was observed in almost all the animals given 300 or 1000 mg/kg/day from day 5, the body weight gain and food consumption were slightly and transiently lower than in controls during the first week. There were no remarkable necropsy findings. In the group given 1000 mg/kg/day, the liver weight was approximately 25% higher than in controls.

- 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.

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

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 sacrifice. The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period, during pregnancy for the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval days 1-5 p.p..

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
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (groups 1 and 4)

Litter observations:

STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after the end of the pairing period (at least 5 weeks of treatment in total),
- Female animals: all surviving animals = day 6 post-partum.
The following F0 females were sacrificed by the same way without overnight fasting:
- females which did not deliver: on day 24 or 25 p.c. (after a body weight recording to check for a possible un-noticed delivery).

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all F0 animals

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- kidneys, cecum (males only), mesenteric lymph nodes, thymus and liver from the low- and intermediate dose groups,
- immunostained kidneys from the control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period,
- reproductive organs from females that did not deliver (control female, low-dose female, mid-dose female and high-dose female).

Postmortem examinations (offspring):

SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups sacrificed on day 5 p.p..

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

yes

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating 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):

Treatment-related clinical signs consisted of ptyalism and piloerection. Ptyalism was recorded in all test item-treated groups with a dose-related increased incidence, and piloerection was recorded with a very low incidence.
Other clinical signs were recorded but none of them were attributed to the test item treatment in view of the lack of dose-relationship (soft feces in 1 male from the low dose group) or they are commonly observed in rat of this strain when housed in laboratory conditions (cutaneous lesions, hair loss).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths in males during the study.
One female given 300 mg/kg/day and one female given 1000 mg/kg/day were sacrificed on day 22 p.c. for ethical reasons (difficulties to deliver). Coldness to the touch, piloerection, round back, pallor of extremities, emaciated appearance and/or soiled urogenital area were noted in both females before sacrifice. Ptyalism was also observed in both females throughout the gestation period. No histopathological examination was carried out in these animals.
One control female and females given respectively 100, 300 and 1000 mg/kg/day were sacrificed on day 24 or 25 post-coïtum (p.c.) in absence of delivery (one female was declared pregnant). Histopathological examination was carried out in these animals. There were no other unscheduled deaths in females during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
There were no effects on mean body weight and mean body weight change in males during the premating period. When compared to the mean values at the end of the dosing period, the body weight gain was 19% lower in the group given the highest dose-level. However this difference was not considered to be of toxicological importance since it was not statistically significantly different and the terminal mean body weight of animals was only 4% lower than that of the control mean value (454 vs. 472 g).

Females
There were no effects on mean body weight and mean body weight change in females during the premating period.
A statistically significant lower mean body weight change was noted in females given 1000 mg/kg/day over the gestation period (-13% vs. controls). However, this variation was not considered to be of toxicological importance as the mean body weight recorded on day 20 of gestation was not statistically significantly different from control and none of the individual values of body weight were below the lowest value of historical control data (306 g on day 20 of gestation).
There were no effects on mean body weight change in females during the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects on mean food consumption during the study

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):

When compared with controls, there was a decrease in white blood cell count (mainly due to decrease of lymphocytes and eosinophils) in the high dose group (males only). In both genders, values of APPT were shortened compared to control values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

When compared with the mean control values, the cholesterol concentration of males and females treated at 300 and 1000 mg/kg/day was 1.8- to 2.8-fold higher, respectively.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day and in both sexes, the mean urinary volume was higher than that of the control group.
There was a tendency towards a decrease of specific gravity among this group.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Premature sacrifices
Three females (0 mg/kg/day), (100 mg/kg/day) and (300 mg/kg/day) were in diestrus. There were no obvious microscopic abnormalities in the reproductive organs.
Microscopic examination of the reproductive organs in one female (1000 mg/kg/day) showed evidence of a recent pregnancy (large pale corpora lutea, presence of sloughed mucified cells in the vaginal lumen). There was mixed inflammatory infiltrate in the uterine mucosa.

Terminal sacrifice
In males, a careful qualitative histopathological examination of the testes was performed. There were no effects on the germ cells or interstitial cells detected. A single male at 1000 mg/kg/day showed minimal, focal tubular degeneration/atrophy. This isolated finding was considered to be incidental and unrelated to the test item in view of its low magnitude.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects on estrus cycle.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no effects on epididymal sperm count, motility and morphology.
There were no effects on testicular sperm count and daily sperm production rate.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item-related effects on mating and fertility data.
There were no dose-related effects on the mean numbers of corpora lutea, implantations or pups at any dose level, nor on the duration of gestation or the extent of post-implantation losses. The statistical difference calculated for the length of gestation was considered not to be meaningful as the mean value of 21.9 days was due to high proportion of females having 22 days of gestation (vs. 21 in the other groups). This value was also close to the upper limit of historical control data (21.7 days).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes in the kidneys associated with increased in the urine volume, the liver and thymus histopathological findings in both genders at 1000 mg/kg/day

Target system / organ toxicity (P0)

Critical effects observed:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None clinical signs considered to be test item related (comparable incidences in control pups or not dose-related).

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no effects on pup viability.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of treatment with the test item on mean body weight and mean body weight change in pups. Since all mean values were within the historical background data.

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:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no changes at necropsy of dead pups which were indicative of a test item-related effect.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Clinical signs

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Ptyalism		5	9	10			5 P 7G 5L	all (P+G+L)
Piloerection								1L

P: premating and mating periods, G: gestation period; L: Lactation period.

 

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 and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 100, 300 or 1000 mg/kg/day.

Based on the experimental conditions of this study, the following conclusions could be drawn:
- the dose-level of 300 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental and systemic toxicity (the following findings observed at 1000 mg/kg/day being considered as adverse: changes in the kidneys associated with increased in the urine volume, the liver and thymus histopathological findings in both genders). It was worth noting that the nature of the histopathological changes on kidneys in males (hyaline droplets) are considered to be of no toxicological relevance for human risk assessment,
- the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) and for toxic effects on progeny.

Executive summary:

The objective of this 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, during mating and, for females, through gestation until day 5 post-partum (p.p.). This OECD 422 study provides information on the possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a subacute period of time. It can also indicate effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition and on early post-natal development of the pups.

 

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 as an emulsion in the vehicle, sesame oil, at dose-levels of 100, 300 or 1000 mg/kg/day.

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 concentrations of the dose formulations were checked in study weeks 1, 3 and 5.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and at least once daily for clinical signs during the treatment period. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs, abnormal behavior and external abnormalities and weighed on days 1 and 5 p.p.. At the end of the treatment period, Functional Observation Battery (FOB), motor activity and laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on five males and five females from each group. Males were sacrificed after completion of the mating period and dams were sacrificed on day 6 p.p.. Epididymal and testicular sperm parameters were evaluated. Final body weights and selected organs weights (adrenals, brain, epididymes, heart, kidneys, liver, ovaries (with oviducts), spleen, testes, thymus, thyroids with parathyroids and uterus (horns and cervix)) 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 selected organs from the first five males sacrificed at the end of the treatment period and the first five females to deliver in the control and high-dose groups, on all macroscopic lesions, on kidneys, cecum (males only), mesenteric lymph nodes, thymus and liver from the low- and intermediate dose groups, and on reproductive organs from females that did not deliver.In addition, immunostaining againsta 2u globulin was performed on kidneys from the first five males sacrificed at the end of the treatment period in the control and high-dose group. Pups were sacrificed on day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

Results

 

The test item concentrations in the dose formulations analyzed in weeks 1, 3 and 5 were within the acceptable range of variation (± 15%).

There were no test item-related deaths.Ptyalism was the only recorded clinical sign, it was observed in all test item-treated groups with a dose-related increased incidence. Only one lactating female displayed piloerection at 1000 mg/kg/day.

There were no toxicologically relevant effects on mean body weight, mean food consumption, mean FOB and motor activity data in any group and sex.

There were no effects on sperm motility, morphology and count and, on testicular sperm head count and daily sperm production rate. There were no toxicologically significant effects on estrus cycle.

There were no macroscopic findings attributed to the test item administration.

 

There were no toxicologically relevant effects on mean mating, fertility and delivery data in any group. There were no test item-related effects on pups by day 5 post-partum (viability, clinical signs, sex ratio, macroscopic post-mortem findings).

Conclusion

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

Based on the experimental conditions of this study, the following conclusions could be drawn:

- the dose-level of 300 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for parental and systemic toxicity (the following findings observed at 1000 mg/kg/day being considered as adverse: changes in the kidneys associated with increased in the urine volume, the liver and thymus histopathological findings in both genders). It was worth noting that the nature of the histopathological changes on kidneys in males (hyaline droplets) are considered to be of no toxicological relevance for human risk assessment,

- the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) and for toxic effects on progeny.