4-(4-isopropoxyphenylsulfonyl)phenol

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-27 to 2009-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

1983-05-26

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance no. 43 on Mammalian Reproductive Toxicity Testing and Assessment; 2008-07-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(4-isopropoxyphenylsulfonyl)phenol

EC Number:

405-520-5

EC Name:

4-(4-isopropoxyphenylsulfonyl)phenol

Cas Number:

95235-30-6

Molecular formula:

C15H16SO4

IUPAC Name:

4-[4-(propan-2-yloxy)benzenesulfonyl]phenol

Details on test material:

- Name of test material: D-8;
- Molecular formula: C15 H16 O4 S;
- Molecular weight: 292.3;
- Physical state: powder,
- Analytical purity: 99.5 %;
- Purity test date: 2008-09-17;
- Lot/batch No.: IHI-006;
- Expiration date of the lot/batch: 2011-09-12
- Stability under test conditions: 36 months;
- Storage condition of test material: Keep away from direct sunlight. Store in a cool dry place. Keep away from oxidizing agents, and high temperature.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: Young adult rats, males approximately 5-6 weeks (15-16 weeks at mating) and females: approximately 8-9 weeks (12-13 weeks at mating)
- Weight at study initiation: The weight variation in animals involved in the study did not exceed > 20 % of the mean weight for each sex; 153-212 g, males, 210-261 g, females
- Fasting period before study: No;
- Housing: Before mating: 4 animals of the same sex/ cage; Mating: 1 male and 1 female / cage; Pregnant females were housed individually; the bedding material was suitable for nesting
- Diet: Animals received ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice – breeding and maintenance” produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water, as for human consumption, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.2 - 24.7 °C
- Humidity: 30 - 70 % R.H.
- Air changes: 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 1.25, 6.25 and 31.25 mg/mL prepared in PEG 400, corresponding to 0, 5, 25 and 125 mg/kg bw/day dose levels, at a 4 mL/kg bw treatment volume.

VEHICLE
- Justification for use and choice of vehicle : Polyethylene glycol 400 (PEG400), Ph. Eur was used (Standard vehicle)
- Lot/batch no.: 1421464, 1403539

Details on mating procedure:

- M/F ratio per cage: Mating began approximately 10 weeks after the initiation of treatment in the male animals, and approximately 4 weeks after the initiation of treatment in the female animals, with one female and one male of the same dose group (1:1 mating) in a single cage.
- Length of cohabitation: Females remained with the same male until copulation occurred (up to 6 days).
- Proof of pregnancy:
Each morning from the onset of treatment of the female rats until mating was completed, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. Assessment of the female oestrus cycle was performed; the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy or gestation day GD0). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC system: Merck-Hitachi LaChrom HPLC system
Assessment of D-8 stability in PEG 400 vehicle at concentrations from approximately 1 to 250 mg/mL indicated an up to 24-hour stability at room temperature, and an up to 72-hour stability when stored refrigerated at 5±3 °C, with a recovery within the acceptable range of 100 ± 10% (96-102 %, and 99-104 %, respectively). All dose formulations were homogenous at all analytical occasions. No D-8 was detected in the vehicle control samples. In the test item samples, the measured concentrations
ranged from 97 to 102% of nominal concentrations (1.25, 6.25 and 31.25 mg/mL).

Duration of treatment / exposure:

Daily dosing of the parent (P) males began approximately 10 weeks prior to the mating period and continued during the mating period until termination. Daily dosing of the parent (P) females began 4 weeks prior to the mating period and continued during the mating period, throughout pregnancy and up to the weaning of the F1 offspring (until termination on postpartal PND 21, or shortly thereafter). Males were dosed during growth and for at least one complete spermatogenic cycle (at least 70 days) in order to elicit any adverse effects on spermatogenesis by the test item. Females of the P generation were dosed for at least two complete oestrous cycles in order to elicit any adverse effects on oestrus by the test substance. The animals were then mated. The test item was administered to both sexes during the mating period and thereafter only to females during pregnancy and for the duration of the nursing period.

Frequency of treatment:

7 days/week, at similar times in the morning

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

125 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 males/24 females per dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on available information from repeated dose toxicity studies with the test item. Based on the NOEAL of 50 mg/kg bw/day in the 90-day study the highest dose level for the one-generation study in the same strain and using the same administration way was established to be 125 mg/kg bw/day in order to induce an acceptable parental toxicity. Based on the results of the 28-day feeding study (see dose selcetion rationale of the 90-day study) higher doses were expected to exceed the maximum tolerable dose (MTD).
- Rationale for animal assignment: random

The following dosing scheme was used:

P generation:
a. Males:
A: Acclimatisation period at least 5 Days;
PM: Pre-mating period at least 70 Days;
M: Mating period up to 21 Days;
-> Necropsy;

b. Females:
A: Acclimatisation period at least 5 Days;
PM: Pre-mating period approximately 4 weeks;
M: Mating period up to 21 Days;
G: Gestation period 22-24 Days;
-> Delivery;
PP/PN (Postpartal/Postnatal Days): Lactation period 21 Days;
-> Necropsy;

Offspring F1 generation:
All F1 offspring were terminated after weaning (postnatal day PND21).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each day) for signs of morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, unusual respiratory pattern), occurrence of secretions and excretions, circulatory and central nervous system, somatomotor activity and behaviour pattern (clonic or tonic movements, stereotypies, bizarre behaviour), changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma; no such clinical signs were noted during the study. Behavioural changes, signs of difficult or prolonged parturition and any signs of toxicity including mortality were paid attention to; no such changes were observed during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: All parent animals were weighed with accuracy of 1 g. Parent animals were weighed on the first day of dosing (Treatment Day 0), at least weekly thereafter and at termination. Parent females were weighed on gestation Days (GD) 0, 7, 14 and 21 and on post partal/natal Days (PND) 0 (within 24 hours after parturition), 7, 14 and 21, and at termination. The body weight of the female animals was additionally measured on GD and PND 4, 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was determined weekly by reweighing the non-consumed diet with accuracy of 1 g during the pre-mating period and during the gestation period. After parturition, the non-consumed diet was weighed on the days when the pups were weighed (PND 0, 4, 7, 14 and 21).

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

Each morning from the onset of treatment of the female rats until mating was completed, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. Assessment of the female oestrus cycle was performed; the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy or gestation day GD0).

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, identified and weighed individually within 24 hours of parturition (on the first day after parturition is complete), then on PND 4, 7, 14, and 21, with an accuracy of 0.1g. Any pup found dead was subjected to necropsy with macroscopic examination. All observed abnormalities were recorded. All the litters were checked and recorded daily for the number of viable and dead pups.

Developmental landmarks were examined daily on each pup of a litter until the respective sign is positive in all the pups of the litter.
- Evidence of suckling was evaluated as of PND0, based on the presence of the milk in pup’s stomach.
- Pinna unfolding: as of PND1, each pup in a litter was examined individually until all pups in the litter meet the criterion for pinna unfolding (i.e. the first day that the point of a pinna/earflap was detached from a rodent pup’s head). The sign was considered positive when at least one pinna was detached.
- Hair growth (first coat): as of PND1, rat pups were examined for appearance of fur on the dorsal surface of the body.
- Surface righting reflex was evaluated as of PND1. It consists of regaining the normal position after the pup is placed on its back, and it is a complex coordinated action that requires many different muscles of the neck, trunk and limbs. Pups were placed on their backs on a flat surface and quickly released. The criterion was considered met when the pup regained its normal position on 4 paws within 5 sec.
- Incisor eruption was evaluated as of PND7. The criterion was considered met when a least one incisor (upper or lower) was visible.
- Negative geotaxis reflex: as of PND7, rodent pups were tested for the ability to change from a downward to an upward orientation on an inclined plane. Each pup was placed facing downhill on a platform at approximately 30°. The sign was considered positive when the pup turned approximately 180° to face completely uphill (body and head) within approximately 60 sec.
- Eye opening: as of PND10, each pup in a litter was examined individually until all pups in the litter met the criterion for eye opening. The sign was considered positive when at least one eye was open (there was a break in the membrane connecting the upper and lower eyelids).
- Auditory startle reflex was evaluated as of PND10, as an observable whole body response (i.e. flinching, jumping, freezing of activity etc) following an auditory stimulus performed in a quiet environment.
- Nipple retention: as of PND11 through 13, all males in each litter were examined for the presence of areola and/or nipples, which should have no longer been present by PND12 or 13. Each pup were carefully examined for the presence of areola and/or nipples (usually only in the thoracic region) by brushing the hair coat.

Postmortem examinations (parental animals):

SACRIFICE
Gross necropsy with macroscopic examination was performed on each parent animal. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia by exsanguination.

GROSS NECROPSY
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormalities were recorded with details of the location, colour, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on the selected list of weighed organs in the control and high dose groups and all macroscopic findings (abnormalities) from the low and mid dose groups. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian
stroma. As no test item related microscopic findings were identified, no additional histology evaluation was considered required.

Paired organs were weighed individually; absolute organ weights were reported. Relative organ weights (to body and brain weight) were calculated and are reported.

At the time of termination, body weight and weight of the following organs of all parental animals were determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain, liver, spleen, kidneys and target organ(s)
- With a precision of 0.001 g: ovaries, pituitary

Postmortem examinations (offspring):

Pups euthanized at PND21 were examined at least externally for gross abnormalities; one male and one female pup randomly selected/litter and all pups found dead were subjected to necropsy with macroscopic examination and retained in formalin for possible future histology evaluation.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was assessed by a Kolmogorov-Smirnov test. In case of a not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If there was a positive result, the inter-group comparisons were performed using a Mann-Whitney U-test. Frequency of toxic response, pathological and histopathological findings and the mean daily food consumption by sex and dose were calculated.

Reproductive indices:

Female fertility index, Gestation index, Female mating index
Male mating index, Male fertility index

Offspring viability indices:

Viability index (%), Lactation index (%), Sex ratio %

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were noted in the male or female animals that could be attributed to D-8 administration by daily oral gavage.
In the female animals, activity decreased, piloerection, pallor and/or hunched back position were noted towards the end of gestation period in 1/24 low dose and 1/24 mid dose females. Based on the low incidence and lack of similar effects noted in the high dose group, these clinical signs are not ascribed to test item administration, but considered correlated with the intrauterine mortality noted in these females which was likely incidental, due to individual variability.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during this study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No effects considered adverse, or toxicologically significant, were noted on the mean body weight and body weight gain values in the treated groups compared to control animals following daily administration of D-8 at dose levels of up to and including 125 mg/kg bw/day. In the male animals, statistically lower mean body weight gain was observed in the high dose 125 mg/kg bw/day group from treatment days 56 to 63 (premating). In the females, the body weight gain was statistically lower during pre-mating (treatment days 14-21), and higher during lactation (days 7-14) in the high dose group.
The absolute body weights compared to the concurrent controls were not significantly reduced; the variations noted in the mean body weight gain values had low incidence and/or magnitude, thus, they were not considered toxicologically significant and/or related to test item administration, which was additionally supported by the absence of any treatment related effects on reproductive performance and on F1 pups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects noted in the food consumption during the study, in both the male and female animals, at dose levels of up to and including 125 mg/kg bw/day.

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:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of D-8-related findings histologically. There were no microscopic findings considered related to treatment in the selected list of organs submitted for histology evaluation, including liver, spleen and kidneys in the High and Control animals, or abnormalities from all groups. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Bone marrow evaluation
There were no test item related effects noted in the examined animals following administration of D-8 in the conditions of this study. Normal haemopoiesis could be observed in all animals. In the control group, four male animals and three female animals showed slightly increased erythropoiesis within physiological range. In the high dose evaluated animals, a 8.6 % increase of myeloid: erythroid ratio was noted compared to the control group in the males (1.16 vs. 1.26), and a 3.2 % increase in the females (1.25 vs. 1.29). These variations were within physiological range. No pathological cells or pathological alteration of normal cell distribution were observed. Variations of the examined parameters were noted, on occasion attaining statistical significance; however, these variations were normal and in the absence of a dose response they were considered incidental, attributed to individual variability, of no toxicological significance and/or not related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item related differences in the oestrous cycle during the four weeks of the pre-mating period. The number of animals with regular or irregular cycles, the number of cycles, the number of days in pro-oestrous, the number of days in oestrous, the number of days in diestrous and number of animals in prolonged diestrous were comparable in the control and treated groups. A relatively high number of animals with prolonged diestrous was observed throughout all groups, including controls, possibly related to pseudo-pregnancies related to repeated collection of vaginal smears.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

In the testes, the spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no D-8 related differences between the control and test item-treated groups with regard to reproductive ability, and in the mating, fertility and gestation indices. The mating index was 100 % in all groups, in both male and female animals. Fertility indices varied from 79 to 96% in all groups; however, as no dose response was noted, and the control animals showed lower values than the treated groups, these variations were considered individual variability and not related to test item administration. No significant changes were observed in the gestation index values.

Details on results (P0)

No test item effect on the duration of pregnancy, or abnormalities in the gestation outcome were observed that could be ascribed to the treatment. The mean duration of pregnancy showed minor variations, not considered biologically significant, from 21.42 days (21 to 22 days) in the controls, 21.43 days (21 to 22 days) in the low dose, 21.32 days (21 to 22 days) in the mid dose, to 21 days in the high dose group (statistically significant, p<0.01). Females were allowed to litter and rear their offspring. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition, or post-partal period noted in the treated groups at dose levels up to and including 125 mg/kg bw/day compared to control animals. All the parturitions were normal. The number of corpora lutea and implantations were comparable in the treated groups up to and including 125 mg/kg bw/day, compared to females in the control groups. There were no statistically significant variations noted in the pre-implantation, intrauterine and post-natal mortality values in the treated groups than in the control animals. Slightly higher pre-implantation, intrauterine and post-natal mortality values without attaining statistical significance were observed in the 25 mg/kg bw/day group compared to control, leading to a statistically higher total mortality in this group, variation likely attributed to female 3512, of whom no pups survived to completion of the study. Other differences were minor, and based on the lack of similar changes in the high dose group, these variations were neither considered toxicologically significant, nor related to test item administration.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no mortality or clinical signs in the preterminally dead pups that could be attributed to D-8 administration. No clinical or behavioural changes were noted in the surviving pups following administration of D-8. On isolated occasions, surviving pups showed haemorrhages on the neck or muzzle (control group), or were cold to touch, but recovered and survived to scheduled termination; these changes were considered incidental and not related to test item administration.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Pup body weight and body weight gain evaluated as litter means on PND 0, 4, 7, 14, and 21 did not show any significant differences, or adverse effects compared to control animals, following administration of D-8 daily by oral gavage, at dose levels of up to and including 125 mg/kg bw/day. Statistically significant lower body weight and body weight gain values were noted when evaluated for all pups, mostly in the low and high dose groups; however, no dose response was observed, the differences were minor and thus not considered related to test item administration.

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

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No retained nipples/areolas were observed in the male pups that could have suggested an adverse, potentially antiandrogenic effect.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Litter examination did not reveal any effects considered directly test item-related compared to observations noted in the control group. No external abnormalities ascribed to test item administration were detected at the clinical or external macroscopic examinations of the pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item related effects were noted on the lactation index. The sex ratios were similar in the control and treated groups. No statistically significant variations were observed on PND4, 7, 14, or 21 compared to PND0. There were no D-8 related adverse effects on the developmental landmarks tested at different timepoints either for all pups, or as mean litter data. The number of animals with positive and negative response showed the normal, expected distribution for each milestone evaluated, although statistically significant variations were noted on occasions due to biological variability.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Summary of reproductive ability results

	Dose (mg/kg bw/day)
	Control	5	25	125
Males
Number of treated males	24	24	24	24
Paired males	24/24	24/24	24/24	24/24
Mated males	24/24	24/24	24/24	24/24
Infertile males	5/24	1/24	1/24	4/24
Male Mating Index (%)	100	100	100	100
Male Fertility Index (%)	79	96	96	83
Females
Number of treated females	24	24	24	24
Paired females	24/24	24/24	24/24	24/24
Mated females	24/24	24/24	24/24	24/24
Sperm-positive females	24/24	24/24	24/24	24/24
Pregnant females	19/24	23/24	23/24	20/24
Delivered dams	19/24	22/24	22/24	20/24
Female Mating Index (%)	100	100	100	100
Female Fertility Index (%)	79	96	96	83
Gestation Index (%)	100	96	96	100

	Dose (mg/kg bw/day)
	Control	5	25	125
Duration of mating period (days)#	1-4	1-5	1-4	1-5
Surviving females showing evidence of copulation (N)	24/24	24/24	24/24	24/24
Females achieving pregnancy (N)	19/24	23/24	23/24	20/24

# from pairing up to successful coitus/sperm positive and/or vaginal plug.

Table 2: Evaluation of Gestation, Parturition and Post-Partal Period

	Dose (mg/kg bw/day)
	Control	5	25	125
Female
Number of treated females	24	24	24	24
Number of delivered dams/dams with implantations	19/19	22/23	22/23	20/20
Duration of pregnancy (days)	21.42	21.23	21.32	21.00
Number of corpora lutea / dams (mean)	15.11	15.09	15.52	14.45
Number of implantations / dams (mean)	15.05	14.48	14.70	14.00
Number of dams with live pups day 0	19	22	22	20
Number of dams with live pups day 4	19	22	21	20
Number of dams with live pups day 21	19	22	21	20
Pre-implantation mortality (%)	0.40	4.31	4.85	3.63
Intrauterine mortality (%)	5.21	9.64	10.09	3.07
Post-natal mortality (%)	1.08	2.23	6.89	2.65
Total mortality (%)	6.27	11.39	14.21	5.60

Table 3: Summary of Offspring Examination

	Dose (mg/kg bw/day)
	Control	5	25	125
	19 dams	22 dams	22 dams	20 dams
Number of pups born	274	308	318	271
Number of live born	271	306	312	270
Number of dead pups (PND0-4)	6	8	21	8
Number of dead pups (PND4-7)	0	0	1	0
Number of dead pups (PND7-14)	1	0	0	0
Number of dead pups (PND14-21)	0	0	0	0
Total number of dead pups	7	8	22	8
% of dead pups	3	3	7	3

Table 4: Incidence of the microscopic observations in the reproductive organs in Terminal females

	Females
	Control	High Dose
No. of animals examined	24	24
Organ/Observation
Ovary
Cyst, luteal	0	2

Table 5: Incidence of the microscopic observations in the reproductive organs in Control and High Dose Terminal males

		Males
	Control	High Dose
No. of animals examined	24	24
Organ/Observation
Testes
Retention, spermatid, stage IX-XII	2	4
Atrophy/Degeneration, tubular	1	2
Epididymis
Atrophy, ductal	0	1
Sperm content, reduced	0	1
Seminal vesicles
Atrophy	1	1
Prostate
Inflammation, subacute	0	2
Inflammation, chronic	1	2
Congestion and/or Hemorrhage	0	2
Cell debris	1	2

Table 6: P (PARENT) FEMALES: SUMMARY DATA OF ESTRUS CYCLE

		DOSE (mg/kg bw/day)
		Control	5	25	125
Number of animals examined	N	24	24	24	24
Animals with regular cycles	N	12	14	8	18
	%	50	58	33	75
Animals with irregular cycles	N	12	10	16	6
	%	50	42	67	25
Number of cycles	Mean	5.8	6.1	5.5	6.1
	SD	1.74	1.33	1.53	1.51
	N	24	24	24	24
Days in proestrous	Mean	5.9	5.9	5.4	6.4
	SD	1.73	1.44	1.64	0.97
	N	24	24	24	24
Days in oestrous	Mean	5.7	6.0	5.5	6.1
	SD	1.76	1.33	1.53	1.51
	N	24	24	24	24
Days in diestrous	Mean	15.1	15.3	16.3	15.5
	SD	4.26	3.03	2.85	2.60
	N	24	24	24	24
Animals in prolonged oestrous	N	0	1	1	0
	%	0	4	4	0
Animals in prolonged diestrous	N	12	10	15	6
	%	50	42	63	25

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

U = Mann-Whitney U-Test Versus Control

DN = Duncan's Multiple Range Test

Table 7: P (PARENT) MALES: SUMMARY DATA OF REPRODUCTIVE ABILITY

		DOSE (mg/kg bw/day)
		Control	5	25	125
No. of males paired	24	24	24	24
Unmated males	N	0	0	0	0
	%	0	0	0	0
Mated males	N	24	24	24	24
	%	100	100	100	100
Fertile males	N	19	23	23	20
	%	79	96	96	83
Mating index	%	100	100	100	100
Fertility index	%	79	96	96	83

Remarks:

* = p < 0.05 CH2

** = p < 0.01 CH2

Table 8: Summary of the pups viability and sex ratios at different timepoints is presented below:

	Dose (mg/kg bw/day)
	Control	5	25	125
	19 dams	22 dams	22 dams	20 dams
Number of pups born	274	308	318	271
Number of live born	271	306	312	270
PND0
Number of viable pups	271	306	308	267
Male (N)	136	166	148	144
Female (N)	135	140	164	123
Survival/viability index (mean)	98.9	99.4	98.1	99.6
Sex ratio at birth (%)	50	54	47	54
PND4
Number of viable pups	268	300	297	263
Male (N)	135	163	143	142
Female (N)	133	137	154	121
Survival index (mean)	97.8	97.4	93.4	97.0
Viability index (mean)	98.9	98.0	95.2	97.4
Sex ratio at PN4 (%)	50	53	46	53
PND7
Number of viable pups	268	300	296	263
Male (N)	135	163	142	142
Female (N)	133	137	154	121
Survival index (mean)	97.8	97.4	93.1	97.0
Viability index (mean)	100.0	100.0	99.7	100.0
Sex ratio at PN7 (%)	50	54	48	54
PND14
Number of viable pups	267	300	296	263
Male (N)	135	163	142	142
Female (N)	132	137	154	121
Survival index (mean)	97.4	97.4	93.1	97.0
Viability index (mean)	99.6	100.0	100.0	100.0
Sex ratio at PN14 (%)	51	54	48	54
PND21
Number of viable pups	267	300	296	263
Male (N)	135	163	142	142
Female (N)	132	137	154	121
Survival index (mean)	97.4	97.4	93.1	97.0
Viability index (mean)	100.0	100.0	100.0	100.0
Sex ratio at PN21 (%)	51	54	48	54
Lactation index (%)	99	98	96	99

Table 9: Tabular Summary Report of Effects of the Test Item on Reproduction/Development

	Dose (mg/kg bw/day)
	Control	5	25	125
Pairs started (N)	24	24	24	24
Surviving females showing evidence of copulation (N)	24	24	24	24
Females achieving pregnancy (N)	19	23	23	20
Females delivered (N)	19	22	22	20
Conceiving days 1 - 5 (N)	19	22	22	20
Conceiving days 6 - 14 (N)	0	0	0	0
Pregnancy ≤ 21 days (N)	11	17	15	24
Pregnancy = 22 days (N)	8	5	7	0
Pregnancy ≥ 23 days (N)	0	0	0	0
Dams with live young born (N)	19	22	22	20
Dams with live young at PND4 (N)	19	22	21	20
Dams with live young at PND21 (N)	19	22	21	20
Corpora lutea/dam (mean)	15.11	15.09	15.52	14.45
Implantations/dam (mean)	15.05	14.48	14.70	14.00
Live pups/dam at birth (mean)	14.42	13.39	13.83	13.55
Live pups/dam at day 4 (mean)	14.11	13.04	12.91	13.15
Sex ratio at birth (%)	50	54	47	54
Sex ratio at PND4 (%)	50	53	46	53
Pup weight at birth (mean, litter data)	6.8	6.7	6.6	6.7
Pup weight at day 4 (mean, litter data)	11.7	11.5	11.4	11.3
STRUCTURALLY ABNORMAL PUPS
Dams with 0#	19/19	22/22	22/22	20/20
Dams with 1	0	0	0	0
Dams with ≥2	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	18/19	16/22	16/22	16/20
Females with 1	1/19	5/22	5/22	1/20
Females with 2	0/19	1/22	1/22	2/20
Females with ≥3	0/19	0/22	0/22	1/20
Pre-natal/post-implantations (implantation's minus live births) (intrauterine)
Females with 0	12/19	14/22	13/22	13/20
Females with 1	3/19	4/22	4/22	5/20
Females with 2	2/19	1/22	4/22	1/20
Females with ≥3	2/19	3/22	1/22	1/20
Post-natal (live births minus alive at post-natal day 4)
Females with 0	16/19	16/22	17/22	14/20
Females with 1	3/19	6/22	3/22	5/20
Females with 2	0/19	0/22	0/22	1/20
Females with ≥3	0/19	0/22	2/22	0/20

# Dams with 0/Dams with live young born

Applicant's summary and conclusion

Conclusions:

In conclusion, D-8 administered daily by oral gavage to Wistar rats at least during premating and mating periods in male animals and premating, mating, gestation and 21-day lactation periods in female animals did not lead to any toxicologically adverse effects at dose levels of 0, 5, 25 and 125 mg/kg bw/day in either the parent or offspring generation. Under the conditions of this study, the no observed effect level (NOEL) for D-8 for parental and F1 offspring effects is 125 mg/kg bw/day.

Executive summary:

A one generation reproduction oral toxicity study including OECD421 parameters was performed with D-8 in male and female CRL:(WI) BR Wistar rats. A control and three dose groups (n= 24 animals per group and sex) were included in the study. The test item was administered daily by oral gavage, at concentrations of 0, 1.25, 6.25 and 31.25 mg/mL prepared in PEG 400 corresponding to 0, 5, 25 and 125 mg/kg bw/day dose levels at a 4 mL/kg bw treatment volume. Males were dosed during growth and for at least one complete spermatogenic cycle (at least 70 days) in order to elicit any adverse effects on spermatogenesis by the test item. Females of the P generation were dosed for at least two complete oestrous cycles in order to elicit any adverse effects on oestrus by the test substance. The animals were then mated. The test item was administered to both sexes during the mating period and thereafter only to females during pregnancy and for the duration of the nursing period, up to at least 21 days. Stability and homogeneity of test item in this vehicle were analytically proven. Assessment of D-8 stability in PEG 400 vehicle at concentrations of approximately 1 to 250 mg/mL indicated an up to 24-hour stability at room temperature, and an up to 72-hour stability when stored refrigerated at 5±3 °C, with a recovery within the acceptable range of 100 ± 10% (96-102 %, and 99-104 %, respectively).

Analyses of dose formulations (concentration and homogeneity) were conducted on three occasions during the study (first weeks of treatment for males and females, respectively, and last week of treatment of the females. All dose formulations were homogenous at all analytical occasions. No D-8 was detected in the vehicle control samples. In the test item samples, the measured concentrations ranged from 97% to 102% of nominal concentrations (1.25, 6.25 and 31.25 mg/mL). These results were considered suitable for the study purposes.

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). Clinical observations for signs of ill health or reaction to treatment were made once daily. A detailed clinical examination was made before the first treatment, then once weekly during the study with the examinations performed with animals removed from the cage. Oestrus cycle was monitored in the female animals for at least 4 weeks, form the onset of treatment until mating has occurred. Body weight and food consumption measurements were performed at least weekly, or more frequently. Females were allowed to litter and rear their offspring. The delivery process and nesting behaviour were carefully observed. Each litter was examined after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities. Live pups were counted, sexed, identified and weighed individually within 24 hours of parturition (on the first day after parturition is complete), then on PND 4, 7, 14, and 21. Any pups found dead were subjected to necropsy with macroscopic examination. All the litters were checked and recorded daily for the number of viable and dead pups and examined individually for a selected list of developmental landmarks until the respective sign was positive in all the pups of the litter.

Gross necropsy with macroscopic examination was performed on each parent animal, with special attention on the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded. Terminal body weight and weights of selected list of organs (uterus, with and without cervix, vagina, testes, epididymides, total and cauda, prostate, seminal vesicles with coagulating glands, brain, liver, spleen and kidneys) were measured, and bone marrow smears were prepared, then examined for the control and high dose group animals, with the exception mentioned in the Deviations to the Study Plan section.

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved, testes and epididymides in Bouin’s solution, all other organs in 10% buffered formalin solution. Pups euthanized at PND21 were examined at least externally for gross abnormalities; one male and one female pup that were randomly selected/litter and all pups found dead were subjected to necropsy with macroscopic examination and retained in formalin for possible future histology evaluation. Detailed histological examination was performed on the selected list of weighed organs in the control and high dose groups and on all macroscopic findings (abnormalities) from the low and mid dose groups. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. As no test item related microscopic findings were identified, no additional histology evaluation was required.

Results and Discussions

D-8 administered daily by oral gavage in Wistar rats in the conditions of this study, at 5, 25 and 125 mg/kg bw/day in PEG400 did not result in any clinical changes that could be ascribed to the treatment. No effects considered adverse, or toxicologically significant, were noted on the mean body weight, body weight gain, or food consumption values in the treated groups compared to control animals.The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, gestation, parturition or post-partal period. There were no adverse findings at macroscopic or microscopic examination at up to and including 125 mg/kg bw/day. There were no treatment-related organ weight changes, or adverse effects observed at bone marrow smears evaluation. In the F1 generation, there were no pups with adverse clinical or developmental changes that could be ascribed to test item administration, or gross abnormalities. No toxicologically significant effect on pup body weight, or pup survival was noted. In conclusion, D-8 administered daily by oral gavage to Wistar rats at least during premating and mating periods in male animals and premating, mating, gestation and 21-day lactation periods in female animals did not lead to any toxicologically adverse effects at dose levels of 0, 5, 25 and 125 mg/kg bw/day in either the parent or offspring generation. Under the conditions of this study, the no observed effect level (NOEL) for D-8 for parental and F1 offspring effects is 125 mg/kg bw/day.