6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the result obtained, the study indicated that dosages of 62.5, 250, or 1000 mg/kg/day were without adverse effect on the growth and reproductive capacity of male and female rats or the development of their offspring. The dosage of of the test substance at which no signs of toxicity were recorded is therefore considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-10-05 to 1993-03-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1983-05

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD (SD) BR VAF/Plus strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) male: 4 wks, female: 7 - 8 wks
- Weight at study initiation: (P) Males: 72 - 95 g; Females:174 - 196 g
- Housing: During the pre-mating period, males and females were housed separately, four to a cage. During the mating period, animals were housed on the basis of one male to one female in plastic breeding cages (North Kent Plastics, RM-2 type). At the end of the mating period the males were rehoused with their former cage mates in metal cages (Biotech®) and the females remained in individual breeding cages (North Kent Plastics, RM-2 type) for the birth and rearing of young. Following the sacrifice of weanlings, females were rehoused with their former cage mates in metal cages (Biotech®).
- Diet: Biosure Laboratory Animal Diet No. 1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 45 ± 17
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 1 % methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 1 mL/100 gram bodyweight
The test substance was ground in a mortar with a small amount of the vehicle (1 % methylcellulose) until a smooth paste was formed. The formulation was then gradually made up to volume and mixed using a high shear homogeniser to give the concentration used for the highest dosage (10.0% w/v). A sequence of suspensions was then made from the highest concentration to give concentrations of 2.5 and 0.625% w/v for the lower dosages. A constant dose volume of 1 mL/100 g bodyweight was assumed throughout.

DIET PREPARATION
- Rate of preparation of diet: daily

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 20 days
- Proof of pregnancy: Sperm in vaginal smear.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Treatment commenced when the males were approximately 6 weeks of age (designated time 0 with the succeeding week designated Week 1 of the study) and continued for 10 weeks prior to mating, through the mating, gestation and lactation periods and up to sacrifice after weaning of pups. The dose volume was calculated for individual animals on the first day of treatment and adjusted at weekly intervals throughout the study.
Sexually mature females were treated for 2 weeks prior to mating (treatment commenced after the males had been treated for 8 weeks). Dosing continued throughout mating, gestation and lactation and up to sacrifice after weaning. The dose volume was calculated for individual animals on the first day of treatment and adjusted at weekly intervals up to and including the second week after pairing. Dose volumes remained constant for each animal thereafter until at least 7 days after Day 21 post partum when volumes were readjusted at weekly intervals up to sacrifice.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

62.5 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and examined for obvious changes or signs of reaction to treatment.

BODY WEIGHT: Yes
- Time schedule: During the mating period, all females were weighed daily throughout and daily weighing continued until parturition. Weights are reported for Days 0, 7, 14, 17 and 20 of pregnancy. Weights of pregnant animals without a positive indication of mating are reported for appropriate days taken retrospectively from birth. Dams that littered were weighed on Days 0, 7, 14 and 21 post partum.

FOOD CONSUMPTION:
- Food consumption: Yes
- Time schedule for examinations: Food intake of rats was recorded weekly for the week inunediately prior to treatment and throughout the pre-mating phase. During the pre-mating phase, food conversion ratios were calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured on a daily basis during the pre-dose week for both sexes, during the first two weeks of treatment for males and for the two weeks prior to mating (males and females). Water consumption was also recorded from allocation until initiation of treatment (Week -1 for males, Week 8 for females).

OTHER:
- Prior to treatment and scheduled terminal sacrifice, all surviving animals were subjected to an ophthalmoscopic examination.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
- Offspring surface righting reflex, offspring startle reflex, offspring air righting reflex, offspring pupil reflex

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed: prostate, seminal vesicles, coagulating gland, epididymides, testes, pituitary, uterus, cervix, vagina, ovaries, oesophagus
The following tissues were additionally weighed: brain, thymus, heart, lungs, liver, kidneys, adrenals

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 21 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

All statistical analyses were carried out separately for males and females.
Significance tests, employing analysis of variance following by an intergroup comparison with control, were performed on the following parameters and results are presented in relevant tables of the report: weekly bodyweight and female bodyweight change during pregnancy and lactation, food and water consumption, litter data and organ weights. Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran, 1967) followed by William’s’ test (Williams, 1971/2)) or non-parametric tests (Kryskal-Wallis (Hollander and Wolfe, 1968) followed by Shirley’s test (Shirley, 1977)) were used to analyze these data, as appropriate. For bodyweight and bodyweight change, the analyses were carried out using the individual animal as the experimental unit. Data relating to food and water consumption were analyses on a cage basis. For litter data, the basic sample unit was the litter and, due to the preponderance of non-normal distributions, nonparametric analyses were routinely used. Organ weight data were analyzed using body weight at post mortem as covariate, to allow for differences in bodyweight which may have influenced organ weight values. Where 75% or more of the values for a given variable were the same, a Fisher’s exact test (Fisher, 1950) was used, when considered necessary. All significant (i.e. P < 0.05) intergroup differences from the control are reported and were supported by a significant analysis of variance (P < 0.05).

Reproductive indices:

Pregnancy rate was determined as the percentage of surviving paired females that became pregnant.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs observed during the study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the high dose group died in week 11 without previous clinical signs. The pathological examination revealed moist red staining of the perioral and perinasal fur and severely congested, uncollapsed lungs, but no signs of an intubation error. One male of the high dose group was sacrificed in week 13 after having been observed continually leaning to one side for nine days. Prior to sacrifice the animal appeared uncoordinated and continually rolled over. No abnormalities were detected during macroscopic post mortem examination.
One female in the low dose group was found dead in week 14, with red salivation and vaginal discharge. No previous clinical signs were recorded. Post mortem findings included enlarged cervical lymph nodes, congested thymus and lungs, gaseous distension, and blood in the stomach, as well as dark contents in the small intestine. The uterus contained recently dead foetuses and placentae. Thirteen pups had been born, all were cold and unfed when the dam was found.
As there were no trends or consistent pathology findings among these animals, the mortalities were considered to be coincidental and not treatment related.
Three female rats, one from control, two from the high dose group, were either found dead or were sacrificed an the second day of treatment following loss of body tone and laboured respiration. Post mortem examination revealed all three to have perforated oesophagus. The clinical signs and necropsy findings were consistent with accidental intubation errors occurring during the dosing procedure. As one of the animals receiving the high dose was found dead within 24 h of the first dose, a replacement animal was selected for the duration of the study from this day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Weekly bodyweight gain for males and femals was generally similar for all groups (P > 0.05).
Bodyweight change for femals during pregnancy and lactation was uneffected by treatment (P > 0.05).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for both sexes recorded during the pre-mating period (to week 10) was essentially similar in all groups (cumulative intake P > 0.05).

Food efficiency:

no effects observed

Description (incidence and severity):

The efficieny of food utilisation during the pre-mating period, although slightly variable, was unaffected by treatment.

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

no effects observed

Description (incidence and severity):

Intergroup variation in water consumption during the periods recorded for males and females showed no dose relationship and cumulative values did not attain statistical significance (P > 0.05).

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The type of ocular lesions seen were consistent with the age and strain of rat. There were no treatment-related changes.

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:

no effects observed

Description (incidence and severity):

No microscopic findings were detected in the tissues examined.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, as assessed by the incidence and distribution of successful matings, the median pre-coital time and the type of smear recorded on the day of conception, was not adversely affected by treatment. The pregnancy rate was 100 % for all groups with most females conceiving within the first four days after the start of the pairing, corresponding with the expected length of an oestrus cycle. The duration of the pregnancy was similar for all groups (P > 0.05).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed up to the highest dose

Key result

Critical effects observed:

no

Mortality / viability:

no mortality observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Intergroup differences in organ weights, adjusted for final bodyweight as appropriate, were only slight (P>0.05) and revealed no clear or consistent changes in either sex which could be attributed to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed at terminal autopsy revealed no treatment-related changes.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mean ages of attainment of surface and air righting reflexes, startle response and presence of pupil reflex at day 20 post partum were similar for treated and control groups.

Two females (one each at 62.5 and 250 mg/kg/day) resorbed their single implant. A single implantation is atypical and is generally considered insufficient to maintain pregnancy thus, in the absence of similar occurrences at the high dose level, the incidence was considered not to be treatment-related.
Among dams rearing young to weaning, mean values for implantation rates, pup survival, pup growth and associated sex ratio at birth and Day 21 post partum were similar for all groups (P>0.05).

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed up to the highest dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Table 1: Summary of Adult Performance

Category	No of Animals in Group/Dosage [mg/kg bw/day]
	Control	62.5	250	1000
Males
Initial Group Size	24	24	24	24
Mortality at Day 19 of Pairing	-	-	-	1**
Inducing Pregnancy	23*	24	24	23 *,**
Females
Initial Group Size	24	24	24	24
Mortaliy:
Pre-Pairing	1	-	-	1
Day 3 of Pairing	-	-	-	1
Day 1 post partum	-	1	1	-
Rearing young to weaning	23	22	23	22
* One male not paired as female partner previously killed ** Male had induced pregnancy prior to sacrifice

Table 2: Group mean Bodyweight Values [g]

Week	Group/Dosage [mg/kg bw/day]
	Male				Female
	Control	62.5	250	1000	Control	62.5	250	1000
-1	137	138	139	138
0 – T male	203	202	204	203
1	264	263	266	261
2	322	321	324	318
3	369	371	373	363
4	406	410	412	400
5	439	445	449	434
6	467	472	480	462
7	492	498	506	486	221	220	220	222
8 – T female	514	520	531	510	244	246	242	243
9	523	532	542	521	255	259	255	256
10 P	541	550	561	537	274	276	272	277
11	548	557	570	550	317	324	315	317
12	566	573	587	564	359	364	358	361
13	574	576	594	566	435	436	432	440
14	588	591	603	577	356	359	357	367
15	606	608	620	590	381	381	381	375
16	621	625	641	604	367	365	360	368
17	634	639	654	617	332	336	327	336
18					337	343	332	341
19					347	354	343	354
20					351	358	350	358
21					355	361	353	361
Bodyweight gain [g/rat]
Weeks Male: 0 – 17 Female: 8 - 21	431	437	451	415	111	115	111	118
S.D.	78.5	52.4	77.9	63.4	15.1	19.8	14.9	19.2
% Control	-	101	105	96	-	104	100	106
S.D. Standard Deviation T Start of treatment P Animals paired

Table 3: Group mean Food Consumption Values [g/rat/week]

Week	Group/Dosage [mg/kg bw/day]
	Male				Female
	Control	62.5	250	1000	Control	62.5	250	1000
-1	154	152	155	151
1 – T male	178	176	173	170
2	199	197	196	190
3	205	205	204	196
4	204	207	209	200
5	208	210	212	201
6	212	212	215	205
7	207	212	212	206
8	204	206	206	202	146	148	148	145
9 – T female	206	209	206	203	147	148	150	144
10	205	209	207	201	152	150	150	149
Cumulative intake [g/rat]
Male: 1 – 10 Female: 9 – 10	2026	2043	2042	1975	299	298	301	294
S.D.	49.5	67.3	103.3	58.6	14.1	13.6	7.6	8.0
% Control	-	101	101	97	-	100	101	98
S.D. Standard Deviation T first week of treatment

Table 4: Group mean Food Conversion Ratio Values (= food consumption/bw gain)

Week	Group/Dosage [mg/kg bw/day]
	Male				Female
	Control	62.5	250	1000	Control	62.5	250	1000
1 – T male	2.9	2.9	2.8	2.9
2	3.4	3.4	3.4	3.3
3	4.3	4.1	4.2	4.3
4	5.6	5.2	5.4	5.4
5	6.2	6.1	5.7	6.0
6	7.8	7.7	7.1	7.1
7	8.2	8.0	7.9	8.6
8	9.3	9.3	8.3	8.5
9 – T female	22.1	18.3	18.4	18.7	13.0	11.6	11.3	10.7
10	11.4	11.7	11.1	12.7	7.7	8.9	8.7	7.1
Mean values
Male: 1 – 10 Female: 9 – 10	6.0	5.9	5.7	5.9	9.7	10.0	9.9	8.5
T first week of treatment

Table 5: Group mean Water Consumption Values [g/rat/week]

Week	Group/Dosage [mg/kg bw/day]
	Male				Female
	Control	62.5	250	1000	Control	62.5	250	1000
-1	184	177	189	183
1 – T male	206	199	218	209
2	232	229	252	229
8					200	218	215	205
9 T – female	257	265	291	264	190	209	207	199
10	255	261	278	269	200	222	210	212
Cumulative intake [g/rat]
Week 1 - 2	438	428	470	438
S.D.	30.4	21.9	28.7	27.1
% Control	-	98	107	100
Week 9 - 10	512	526	568	533	391	431	416	413
S.D.	59.6	71.0	71.1	48.6	33.1	28.0	32.2	29.5
% Control	-	103	111	104	-	110	106	106
S.D. Standard Deviation T first week of treatment

Table 6: Summary of Ophthalmoscopic Observations

Category	No of Animals in Group/Dosage [mg/kg bw/day]
	Control	62.5	250	1000
Males
Pre-treatment (Week -1)
Examined	24	24	24	24
Vitreous: hyaloid remnants	4	1	3	5
Pre-sacrifice (Week 17)
Examined	24	24	24	23
Lens: posterior capsular opacity	-	-	1	-
Vitreous: hyaloid remnants	1	-	-	-
Retina: apparent hyperreflectivity	1	-	-	1
Females
Pre-treatment (Week 8)
Examined	24	24	24	24
Vitreous: hyaloid remnants	6	9	5	5
haemorrhage	-	1	-	-
Pre-sacrifice (Week 21)
Examined	23	23	24	22
Vitreous: hyaloid remnants	-	1	2	1
Retina: abnormality of optic nerve head	1	-	-	1

Table 7: Group Values of Mating Performance

	Group/Dosage [mg/kg bw/day]
	Control	62.5	250	1000
No of mating pairs	23	24	24	22 d
No conceiving on Day
1	12	9	5	7
2	3	2	10	7
3	2	8 r	8	3
4	6	4	-	2
6	-	-	-	2
7	-	-	-	1
15	-	1	-	-
U	-	-	1 r	-
Total	23	24	24	22
Pregnancy rate [%]	100	100	100	100
Median pre-coital time [days]	1.0	3.0	2.0	2.0
Cell type at conception(from vaginal smear)
S	22	23 r	23	18
C	-	1	-	2
L (Day 1)	1	-	-	-
L	-	-	-	2
U	-	-	1 r	-
Duration of pregnancy [days]
21	3	4	4	2
22	14	16	17	16
23	5	3	2	2
U	1	-	-	2
Mean	22.1	22.0	21.9	22.0
d Excludes one dam found dead on day 3 of pairing r Includes one dam with total resorption U Undetermined Predominant cell type: S Sperm C Cornified epithelial L Leucocyte Table 8: Group mean Values of Litter Data   Group/Dosage [mg/kg bw/day] Control 62.5 250 1000 No of animals rearing young to weaning 23 22 23 22 Implementation sites 16.5 17.7 16.7 17.2 At birth   Implant loss [%] 6.6 5.8 5.2 4.3 Total litter size 15.4 16.7 15.8 16.5 Live litter size 15.3 16.6 15.7 16.4 Pup loss [%] 0.3 0.6 0.5 0.8 Litter weight [g] 98.8 103.6 101.5 106.6 Mean pup weight [g] 6.5 6.3 6.5 6.5 At Day 4   Litter size 15.3 16.0 15.6 16.0 Cumulative loss [%] 0.8 3.8 1.1 2.5 Litter weight [g] 156.4 156.8 160.2 162.4 Mean pup weight [g] 10.5 9.9 10.3 10.2 At Day 8   Litter size 15.2 15.8 15.5 15.8 Cumulative loss [%] 1.1 5.1 1.8 4.0 Litter weight [g] 253.9 252.4 261.0 262.3 Mean pup weight [g] 17.1 16.2 17.0 16.7 At Day 12   Litter size 15.2 15.7 15.5 15.7 Cumulative loss [%] 1.1 5.6 1.8 4.4 Litter weight [g] 369.9 369.5 376.2 374.5 Mean pup weight [g] 24.9 23.9 24.6 24.0 At Day 16   Litter size 15.2 15.7 15.5 15.7 Cumulative loss [%] 1.1 5.6 1.8 4.4 Litter weight [g] 470.7 476.0 479.2 480.4 Mean pup weight [g] 31.8 30.9 31.5 30.8 At Day 21   Litter size 15.2 15.7 15.5 15.7 Cumulative loss [%] 1.1 5.6 1.8 4.4 Litter weight [g] 679.6 678.2 686.7 703.0 Mean pup weight [g] 45.9 43.9 45.1 45.1 Table 9: Group mean Values of the Sex Ratio   Group/Dosage [mg/kg bw/day] Control 62.5 250 1000 No of litter 23 22 23 22 Litter size at birth   Male 7.0 7.8 7.8 7.8 Female 8.3 8.9 8.0 8.7 Total 15.4 16.7 15.8 16.5 % Males 46.0 46.0 49.0 46.9 Litter size at Day 21   Male 7.0 7.4 7.7 7.5 Female 8.2 8.3 7.7 8.2 Total 15.2 15.7 15.5 15.7 % Males 46.2 47.0 49.4 47.1   Table 10: Group mean Values of pre-weaning Development   Group/Dosage [mg/kg bw/day] Control 62.5 250 1000 Duration of pregnancy 22.1 22.0 21.9 22.0 Mean age (days post partum) for attaining   Surface righting 24.3 24.2 24.1 24.3 Startle reponse 34.9 35.0 34.8 34.6 Air righting 37.8 37.6 37.7 37.7 Pupil reflex (day 20 post partum   % succesful 100 100 100 100   Table 11: Absolute Organ Weights   Group/Dosage [mg/kg bw/day] Control 62.5 250 1000 Males, absolute weight Number of animals 24 24 24 23 Bodyweight [g] 628 632 646 612 Brain [g] 2.1 2.2 2.1 2.2 Pituitary [mg] 15.4 15.6 15.4 15.5 Thymus [g] 0.29 0.31 0.30 0.29 Heart [g] 1.91 1.91 1.90 1.80 Lungs [g] 2.01 2.06 2.06 2.00 Liver [g] 26.9 27.2 25.9 26.0 Kidneys [g] 4.83 4.75 4.70 4.63 Adrenals [mg] 57.5 59.6 60.1 57.5 Testes left [g] 1.772 1.827 1.847 1.812 Testes right [g] 1.785 1.826 1.831 1.782 Epididymides left [g] 0.685 0.685 0.677 0.676 Epididymides right [g] 0.712 0.713 0.713 0.696 Sem Ves./prostate/coag. Gland 3.199 3.432 3.232 3.58 Females, absolute weight Number of animals 23 23 24 22 Bodyweight [g] 353 359 350 358 Brain [g] 2.0 2.0 2.0 2.0 Pituitary [mg] 16.0 16.2 16.6 15.8 Thymus [g] 0.35 0.36 0.33 0.33 Heart [g] 1.24 1.29 1.25 1.27 Lungs [g] 1.57 1.54 1.52 1.60 Liver [g] 15.3 15.5 15.4 16.1 Kidneys [g] 2.75 2.75 2.77 2.81 Adrenals [mg] 81.5 81.2 81.1 85.3 Ovaries [mg] 118.6 121.6 122.3 127.4

Conclusions:

Based on the result obtained, this study indicated that dosages of 62.5, 250 or 1000 mg/kg/d were without adverse effect on the growth and reproductive capacity of male and female rats or the development of their offspring. The dosage of the test substance at which no signs of toxicity were recorded is therefore considered to be 1000 mg/kg/d.

Executive summary:

In an 1-generation reproduction study according to OECD guideline 415 (Huntingdon, 1993), the test substance was administered to 24 Sprague Dawley rats/sex/dose by gavage at dose levels of 62.5, 250 or 1000 mg/kg/d.

Each group consisted of 24 male and 24 female rats. Animals from all groups were observed for clinical signs, mortality and changes in body weight, food consumption, water consumption and ophthalmoscopy during the experiment. Furthermore pregnancy rate, mating performance (7 days prior to mating and daily during 20 day mating period), duration of pregnancy and different litter data (offspring surface righting reflex, offspring startle reflex, offspring air righting reflex, offspring pupil reflex) were examined.

After a minimum of 10 weeks of treatment, females were cohabitated with males in a 1:1 (one male to one female) ratio. The number, weight, survivability and mortality of pups were observed during the lactation period. Physical signs of postnatal development were observed daily until the criterion was met.

The animals were subjected to detailed necropsy at sacrifice and specified organs were weighed. Histopathological examination of parents was initially carried out for the preserved organs including gross lesions. For F1 weanlings, macroscopical examinations were also performed.

There were no treatment related clinical signs observed during the study. One female receiving 1000 mg/kg/day was found dead during week 11. Post mortem revealed moist red staining of the perioral and perinasal fur and severely congested, uncollapsed lungs but no apparent signs of intubation error. One male receiving 1000 mg/kg/day was sacrificed week 13. Prior to sacrifice this animal appeared uncoordinated and continually rolled over, however there were no macroscopic abnormalities. One female in the low dose group was found dead in week 14. Post mortem findings included enlarged cervical lymph nodes, congested thymus and lungs, gaseous distension of and blood in the stomach, dark contents in the small intestines. Uterus contained recently dead foetuses and placentae; 13 pups had been born. These mortalities were considered to be coincidental and not related to treatment. A further three animals died from accidental intubation errors. No other effects were observed in parental animals with regard to body weight, food consumption, food conversion ratio, water consumption and ophthalmoscopy.

Mating performance, as assessed by the incidence and distribution of successful matings, the median pre-coital time and the type of smear recorded on the day of conception, was not adversely affected by treatment. The pregnancy rate was 100 % for all groups with most females conceiving within the first four days after the start of the pairing, corresponding with the expected length of an oestrus cycle. The duration of the pregnancy was similar for all groups (P > 0.05).

Two females resorbed their single implant. This incidence was considered not to be treatment related. Among dams rearing young to weaning, mean values for implantation rates, pup survival, pup growth and associated sex ratio at birth and Day 21 post partum were similar for all groups.

The macroscopic examination performed at terminal autopsy of surviving adults and offspring revealed no treatment-related changes. No microscopic findings were detected in the tissues examined.

Based on the result obtained, this study indicated that dosages of 62.5, 250, or 1000 mg/kg/day were without adverse effect on the growth and reproductive capacity of male and female rats or the development of their offspring. The dosage of the test substance at which no signs of toxicity were recorded is therefore considered to be 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study report.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a 1-generation reproduction study according to OECD guideline 415 (Huntingdon, 1993), the test substance was administered to 24 Sprague Dawley rats/sex/dose by gavage at dose levels of 62.5, 250 or 1000 mg/kg/d.

Each group consisted of 24 male and 24 female rats. Animals from all groups were observed for clinical signs, mortality and changes in body weight, food consumption, water consumption and ophthalmoscopy during the experiment. Furthermore pregnancy rate, mating performance (7 days prior to mating and daily during 20 day mating period), duration of pregnancy and different litter data (offspring surface righting reflex, offspring startle reflex, offspring air righting reflex, offspring pupil reflex) were examined.

After a minimum of 10 weeks of treatment, females were cohabitated with males in a 1:1 (one male to one female) ratio. The number, weight, survivability and mortality of pups were observed during the lactation period. Physical signs of postnatal development were observed daily until the criterion was met.

The animals were subjected to detailed necropsy at sacrifice and specified organs were weighed. Histopathological examination of parents was initially carried out for the preserved organs including gross lesions.

For F1 weanlings, macroscopical examinations were also performed.

There were no treatment related clinical signs observed during the study. One female animal receiving 1000 mg/kg/day was found dead during week 11. Post mortem examination revealed moist red staining of the perioral and perinasal fur and severely congested, uncollapsed lungs but no apparent signs of intubation error. One male animal receiving 1000 mg/kg/day was sacrificed in week 13. Prior to sacrifice this animal appeared uncoordinated and continually rolled over, however there were no macroscopic abnormalities found. One female animal in the low dose group was found dead in week 14. Post mortem findings included enlarged cervical lymph nodes, congested thymus and lungs, gaseous distension of and blood in the stomach, dark contents in the small intestines. Uterus contained recently dead foetuses and placentae; 13 pups had been born. These mortalities were considered to be coincidental and not related to treatment. A further three animals died from accidental intubation errors.

No other effects were observed in parental animals with regard to body weight, food consumption, food conversion ratio, water consumption and ophthalmoscopy.

Mating performance, as assessed by the incidence and distribution of successful matings, the median pre-coital time and the type of smear recorded on the day of conception, was not adversely affected by treatment. The pregnancy rate was 100 % for all groups with most females conceiving within the first four days after the start of the pairing, corresponding with the expected length of an oestrus cycle. The duration of the pregnancy was similar for all groups (P > 0.05).

Two females resorbed their single implant. This incidence was considered not to be treatment related. Among dams rearing young to weaning, mean values for implantation rates, pup survival, pup growth and associated sex ratio at birth and Day 21 post partum were similar for all groups.

The macroscopic examination performed at terminal autopsy of surviving adults and offspring revealed no treatment-related changes. No microscopic findings were detected in the tissues examined.

Based on the result obtained, this study indicated that dosages of 62.5, 250, or 1000 mg/kg/day were without adverse effect on the growth and reproductive capacity of male and female rats or the development of their offspring. The dosage of the test substance at which no signs of toxicity were recorded is therefore considered to be 1000 mg/kg/day.

Effects on developmental toxicity

Description of key information

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-04 to 2020-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 189-236 g
- Housing:
Before mating: 1-2 females per cage
1-2 males per cage
During mating: 1 male and 1-2 females / cage
During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum, ssniff® SM R/M " breeding and maintenance autoclavable complete feed (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum, tap water from municipal supply
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 24.1
- Humidity (%): 30 - 52
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in the formulation laboratory of the Test Facility not longer than 3 days before the administration. Formulations were stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore 1% Methylcellulose was used.
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 1908-4485

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Five samples from different places were taken from each concentration. Similarly, five samples were taken from the vehicle (Control group) and analyzed. The suitability of the chosen vehicle for the test item at the intended concentrations was verified up front. WinCon-2 recovery was 104 and 101 % of nominal concentrations at 5 and 100 mg/mL in 1% Methylcellulose, respectively. WinCon-2 proved to be stable at room temperature for 24 hours (mean recovery was 102 % of starting concentration at 5 mg/mL and 103 % at 100 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102 % of starting concentration at 5 mg/mL and 99 % at 100 mg/mL). A separate analytical report provided these results.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/2
- Length of cohabitation: 2 - 4 hours
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestational day 5 to 19

Frequency of treatment:

daily

Dose / conc.:

62.5 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 to 26

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on the results obtained in a study with the same test item 6' -(Dibutylamino)-3' -methyl-2' -phenylaminospiro (isobenzofuran-l(3H),9'-(9H)-xanthene)-3-one) “ODB-2 a study in the rat of reproductive function on one generation by oral administration” performed at Huntingdon Research Centre Ltd. 1993 (Guideline for Testing of Chemicals, No. 415, adopted May 1983.) where the dosages of 62.5, 250 or 1000 mg/kg/day did not lead to any effect on the growth and reproductive capacity of male and female rats or development of their offspring. The dosage of the test item at which no signs of toxicity was recorded was therefore considered to be 1000 mg/kg/day which is the limit dose according to the OECD 414 test guideline.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (signs of morbidity and mortality were checked twice daily)
- Cage side observations included check of behavior and general condition, duration and severity of the clinical signs and observations for signs of morbidity and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 5, 8, 11, 14, 17 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Uterus with cervix and ovaries, organ weight of thyroid glands together with the parathyroid glands

OTHER:
Examination of placental signs: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.

Blood Collection and Determination of Serum Levels of TSH, FT3 and FT4: Blood samples collected for TSH and Thyroid Hormones (FT3 and FT4) measurements were drawn from all sperm positive females from the
sublingual vein in the morning on the day of necropsy within a short timeframe (not exceeding two hours).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

Statistics:

Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually. The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females (no implantation, no corpora lutea), total exclusion
- Disease or death of the dam unrelated to the treatment (total exclusion)
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too. A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.

Historical control data:

The results were compared to the laboratory's historical control data (refer to "Historical control data" in "Overall remarks, attachments").

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related clinical signs observed in the females of all dose groups.
1000 mg/kg bw/day: Clinical signs were only observed in 1 moribund female which was euthanized on GD 16 (1/26; unrelated to the test item).
Refer to Appendix 2.1 ("Experimental summary tables" in "Overall remarks, attachments").

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

62.5 mg/kg bw/day: 1/25 sperm positive females died due to a technical reason (mis-gavage).
1000 mg/kg bw/day: 1/26 females was moribund and therefore euthanized on GD 16 (unrelated to the test item).
Refer to Appendix 1 ("Experimental summary tables" in "Overall remarks, attachments").

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight, body weight gain in the different periods as well as corrected body weight of the females was similar in all groups.
Refer to Appendix 3.1 ("Experimental summary tables" in "Overall remarks, attachments").

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant differences in the food consumption of the dams in the dose groups.
Refer to Appendix 5.1 ("Experimental summary tables" in "Overall remarks, attachments").

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

Endocrine findings:

no effects observed

Description (incidence and severity):

The free thyroid hormone (FT3 and FT4) and TSH levels were similar in all dose groups.
Refer to Appendix 6.1 ("Experimental summary tables" in "Overall remarks, attachments").

There were no effects of treatment with the test item in the control, 62.5 and 1000 mg/kg bw/d dose groups. The mean thyroid weight was statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group, however without a dose response. Hence, it was not attributed to any effect of the test item. Thyroid weight was considered to be not effected by treatment.
Refer to Appendix 7.1 ("Experimental summary tables" in "Overall remarks, attachments").

The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Cyst forming was recorded for the thyroid gland of one control female.
Refer to Appendix 8.1 ("Experimental summary tables" in "Overall remarks, attachments").

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):

There were no effects of treatment with the test item in the control, 62.5 and 1000 mg/kg bw/d dose groups. The mean thyroid weight was statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group, however without a dose response. Hence, it was not attributed to any effect of the test item.
Thyroid weight was considered to be not effected by treatment.
There were no test item related differences in gravid uterine weight among the dosing groups.
Refer to Appendix 7.1 and 4.1 ("Experimental summary tables" in "Overall remarks, attachments").

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A dilated renal pelvis was found in two females each in the 62.5, 250 and 1000 mg/kg bw/day dose groups and one female in the control group. This finding was not attributed to the treatment considering the lack of dose response. Diaphragmatic hernia as a developmental disorder (hence not related to the treatment) was observed in one female in the 1000 mg/kg bw/day group. The uterine horn was filled with fluid to distention in one female in the 1000 mg/kg bw/day group. Considering the low incidence, this was not attributed to an effect of the test item. Blood was found in the uterine horn of one dam in the 62.5 and one in the 250 mg/kg bw/day dose group.
Refer to Appendix 2.1 ("Experimental summary tables" in "Overall remarks, attachments").

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The treatment did not result in histological changes of the thyroid gland in any of the dose groups. Cyst forming was recorded for the thyroid gland of one control female.
Refer to Appendix 8.1 ("Experimental summary tables" in "Overall remarks, attachments").

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no increase indicated in the mean percent of pre- and post-implantation loss (early embryonic, late- and fetal death) at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Early or late resorptions:

no effects observed

Description (incidence and severity):

There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no test item related effect at any dose level.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no test item related effect at any dose level.
Refer to Appendix 1 ("Experimental summary tables" in "Overall remarks, attachments").

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related differences in the body weight of the fetuses.
Refer to Appendix 10.1 ("Experimental summary tables" in "Overall remarks, attachments").

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No effects observed. Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex distribution of the fetuses were not influenced by the treatment.
Refer to Appendix 9.1 and 9.2 ("Experimental summary tables" in "Overall remarks, attachments").

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The number of evaluated litters/fetuses was 25/270, 22/235, 23/242 and 22/227 in the control, 62.5, 250 and 1000 mg/kg bw/day groups respectively. There was no dose related increase in the incidence of growth retardation.
Refer to Appendix 9.1, 9.2 and 10.1 ("Experimental summary tables" in "Overall remarks, attachments").

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

There were no treatment related differences in the absolute or normalized ano-genital distance at any dose level.
Refer to Appendix 10.1 ("Experimental summary tables" in "Overall remarks, attachments").

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was 1/22 litters with malformed fetuses in the 1000 mg/kg bw/day groups (edema and multiply malformed; 0733152/1). There were no statistically significant differences in the incidence of malformations over all dosing groups.
Refer to Appendix 11.1 and 12.1 ("Experimental summary tables" in "Overall remarks, attachments").

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1/25, 1/22, 1/23 and 1/22 litters with malformed fetuses (in the control, 62.5, 250 and 1000 mg/kg bw/day groups).
Control group: Scapula bent; radius bent, humerus slightly bent, thicker, femur short, bent (0617194/1)
62.5 mg/kg bw/day: humerus slightly shorter, radius slightly bent (0831119/5)
250 mg/kg bw/d: Thoracic centrum asymmetric, bipartite, cartilage dumb-bell shaped (0513160/7)
1000 mg/kg bw/day: Scapula bent, Mis developed mandible (agnathia) (0733152/1)
There were no statistically significant differences in the incidence of malformations over all dosing groups.

Refer to Appendix 11.1 and 14.1 ("Experimental summary tables" in "Overall remarks, attachments").

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral malformations were reported.
Refer to Appendix 11.1 and 13.1 ("Experimental summary tables" in "Overall remarks, attachments").

Details on embryotoxic / teratogenic effects:

Fetal examinations

The number of litters with malformations was one in all groups. There was no increase of variations during fetal examinations indicated:

External examination: There was no increase indicated in the incidence of external variations and no effect of treatment with the test item was observed. Body weight retardation of the fetuses (below 2.85 g for males and 2.77 g for females) as well as hyperflexion of the forelimbs (not proved skeletally) were evaluated as external variations. There was no dose related increase in the incidence of growth retardation. The incidence was statistically significantly (p<0.05) lower in the 62,5 mg/kg bw/day group. Hyperflexion of the forelimbs (not proved at skeletal examination) was found in two control fetuses (besides the above discussed fetus No.: 0733152/1 in the high dose). One fetus was found as slightly pale in the 250 mg/kg bw/day group, which was not considered as test item related considering the low incidence. Considering the lack of dose response or the low incidence the above variations were not attributed to the treatment.

Visceral examination: There were no statistically significant increases in the incidence of visceral variations and no effect of treatment with the test item was indicated. The variations/abnormalities were statistically significantly lower (p<0.05) in the 250 mg/kg bw/day group.
Slightly dilated third brain ventricle and lateral ventricles were recorded in the control group (5 fetuses) and high dose (2 fetuses). Hydroureter were found in all groups without a dose response. Hydroureter accompanied with dilated renal pelvis was observed in one single fetus in the 1000 mg/kg bw/day group. A slightly malpositioned kidney was found in one control and one high dose fetus. Slightly smaller kidney was recorded for one control fetus. Considering the lack of dose response or the low incidence the above variations were not attributed to the treatment.

Skeletal examination: There was no statistically significant increase in the incidence of skeletal variations and no effect of treatment with the test item was indicated. Statistically significant decreases were seen sporadically in the dose groups (3 ossified sternebra in the 62.5, incompletely or not ossified pubic or ischia in the 62.5, 250 and 1000, less than 3/3.5 ossified metacarpal/metatarsal in the 62.5 and 1000 mg/kg bw/day groups, each significance p<0.05). It was noticed that the occurrence of skeletal variations in the control group was nearly twice as high as in the dosing groups (18.8 fetuses with abnormalities compared with 7.9 – 14.3 fetuses in the dosing groups). Skeletal variations were mainly observed in 2 litters (0210157, 0617194) with between 83 % and 100 % of the fetuses having at least one variation. Thus, the sporadic decrease in the dose groups is mainly based on the higher incidence of skeletal variations observed in the control group.

Refer to Appendix 11.1 and 14.1 ("Experimental summary tables" in "Overall remarks, attachments").

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.

Executive summary:

WinCon-2 was examined for its possible prenatal developmental toxicity. Groups of 25, 26 and 26 sperm-positive female Han: of Wistar origin rats were treated with WinCon-2 by oral administration daily at three dose levels of 62.5, 250 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% methylcellulose. The treatment volume was 10 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. WinCon-2 in 1% methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 oC) at the concentrations of 5 and 100 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 102 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

 

Results

Evaluated litters

In total, on gestation day 20 there were 25, 22, 23 and 22 evaluated litters in the control, 62.5, 250 and 1000 mg/kg bw/day group, respectively.

 

Mortality, clinical signs, necropsy findings

None of the females died before scheduled necropsy due to the test item and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed.

 

Food consumption, body weight

Food consumption and body weight development were not affected by the test item.

 

FT3, FT4 and TSH level

The test item did not influence the thyroid hormone levels in any of the treated animals.

 

Thyroid weight

There were no test item related differences in thyroid weight among the dosing groups.

 

Histopathology of thyroid glands

The treatment did not result in histological changes of the thyroid gland in any of the dose groups.

 

Intrauterine mortality

Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment.

 

Fetal examinations

There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. The number of litters with malformations was one in all groups. There was no increase of variations during fetal examinations indicated.

 

Conclusion

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant study report.

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

WinCon-2 was examined for its possible prenatal developmental toxicity. Groups of 25, 26 and 26 sperm-positive female Han: of Wistar origin rats were treated with WinCon-2 by oral administration daily at three dose levels of 62.5, 250 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle 1% methylcellulose. The treatment volume was 10 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. WinCon-2 in 1% methylcellulose was stable at room temperature for at least one day and for three days in the refrigerator (5 ± 3 oC) at the concentrations of 5 and 100 mg/mL. Analytical control of dosing solutions was performed during the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 102 and 108 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Blood sampling for determination of thyroid hormones FT3 and FT4 as well as TSH, Caesarean section and gross pathology were performed on gestational day 20. Thyroids were weighed and evaluated histologically. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. External fetal sex was determined by gross pathological examination and compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue alterations). The anogenital distance was measured. In addition, indication of incomplete testicular descent / cryptorchidism was noted in male fetuses. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

 

Results

Evaluated litters

In total, on gestation day 20 there were 25, 22, 23 and 22 evaluated litters in the control, 62.5, 250 and 1000 mg/kg bw/day group, respectively.

 

Mortality, clinical signs, necropsy findings

None of the females died before scheduled necropsy due to the test item and there were no test item related clinical signs recorded in the dose groups. No treatment related necropsy findings were observed.

 

Food consumption, body weight

Food consumption and body weight development were not affected by the test item.

 

FT3, FT4 and TSH level

The test item did not influence the thyroid hormone levels in any of the treated animals.

 

Thyroid weight

There were no test item related differences in thyroid weight among the dosing groups.

 

Histopathology of thyroid glands

The treatment did not result in histological changes of the thyroid gland in any of the dose groups.

 

Intrauterine mortality

Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment.

 

Fetal examinations

There were no test item related adverse effects on the fetal- and placental weight, ano-genital distance, external and visceral development of fetuses. The number of litters with malformations was one in all groups. There was no increase of variations during fetal examinations indicated.

 

Conclusion

Oral treatment of pregnant Han: Wistar rats from gestation day 5 up to day 19 (the day before Caesarean section) with WinCon-2 at the dose levels of 62.5, 250 and 1000 mg/kg bw/day did not cause any maternal effects. Fetal examinations did not reveal any adverse effects on the fetuses.

Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 1000 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 1000 mg/kg bw/day

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on reproductive and developmental toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).