3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2021 - 24 Feb 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]

EC Number:

226-999-5

EC Name:

3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]

Cas Number:

5590-18-1

Molecular formula:

C22H6Cl8N4O2

IUPAC Name:

3,3'-(1,4-phenylenediimino)bis(4,5,6,7-tetrachloro-1H-isoindol-1-one)

Constituent 2

Reference substance name:

mono/bis-methoxy derivatives

Molecular formula:

C22-24H6-12Cl6-8N4O2-4

IUPAC Name:

mono/bis-methoxy derivatives

Test material form:

solid: bulk

Specific details on test material used for the study:

TEST MATERIAL
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g
- Expiry date: 29 December 2027
- Appearance: Solid, orange

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: proven.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspensions of the test item, in 0.5 % CMC, was prepared using the following procedure:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly, since the stability data supported the weekly preparation.


FORM AS APPLIED IN THE TEST: suspension in vehicle

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks (virgin females); 11 weeks (males)
- Weight at study initiation: 200-225 g (females); at least 350 g (males)
- Fasting period before study: not specified
- Housing: no more than 5 of one sex to a cage (before mating for all animals and after mating for males); one male with one female rat (during the mating period); individually (mated females)
- Diet (e.g. ad libitum): commercially available laboratory rodent diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 - 20
- Photoperiod (hrs dark / hrs light): 12 hours

IN-LIFE DATES: From: 2021-05-27 (arrival) To: 2021-07-19 (start of necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1. The required amount of test item was weighed.
2. The required amount of vehicle was added.
3. The mixture was treated with a Silverson (medium head) for approximately 5 minutes.
4. The resulting suspension was left under magnetic stirring for at least 1 hour prior to dosing or analysis. The formulation was prepared daily or weekly.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis were performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity and concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Day 6 through Day 19 post coitum.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 mated female rats/ dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected by the Sponsor.
- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified.
- Time of day for (rat) dam blood sampling: The blood sampling was performed on the morning of the day of necropsy.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the afternoon for mortality. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. Severely debilitated animals were observed carefully. Clinical signs were recorded daily starting from allocation until sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 post coitum
- All animals, including those found dead, were euthanized by carbon dioxide inhalation and subjected to necropsy. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
- Organs examined: From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin.


OTHER:
- Thyroid hormone determination (T3, T4 and TSH): all females, samples taken on day 20 post coitum, determination via immunoanalysis

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead or killed during the study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number, sex and weight of all live foetuses; number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing); gross evaluation of placentae; Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: approximately 1 mL
- Other: On Day 20 post coitum, blood samples for thyroid hormones determination were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short timeframe (e.g. two hours, if possible) on the morning of the day of necropsy. Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.
- Immunoanalysis: Serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH))

Fetal examinations:

- External examinations: Yes: all live foetuses
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes: all live foetuses on Day 20 post coitum

Structural deviations were classified as follows:
- Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation.
- Anomalies: minor abnormalities that are detected relatively frequently.
- Variants: a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Statistics:

For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Indices:

- Corrected maternal body weight (Body weight on Day 20 post coitum minus gravid uterus weight)
- Corrected maternal body weight gain (Body weight on Day 20 post coitum minus gravid uterus weight minus maternal weight on Day 6 post coitum)
- Pre-implantation loss: [(no. of corpora lutea - no. of implantations) x 100] / no. of corpora lutea
- Post-implantation loss: [(no. of implantations - no. of live foetuses) x 100] / no. of implantations
- Total implantation loss: [(no. of corpora lutea - no. of live foetuses) x 100] / no. of corpora lutea
- Sex ratios of the foetuses were calculated as the percentage of males per litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment related adverse clinical signs were described.

Hairloss was observed in one control female, in one mid- dose female and in 2 high dose females.
Considering the low incidence of hariloss and the presence of the sign also in a control animal the observation was deemed representative of normal background variability within the Wistar Han rat.
During the last days of gestation period, yellow faeces were observed in all high dose group females.
One low dose female showed a palpable mass on gestation Days 18-20. The presence of mass is considered spontaneous in origin.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weight and body weight gain were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between groups.

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:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were noted. Lower T4 levels were noted at 1000 mg/kg body weight, but the values were within the historical control data. Furthermore, no changes occurred in T3 and TSH values, thyroid gland weights and histopathological examination of the thyroid gland. Therefore, this difference was considered as incidental.

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):

No changes in organ weights were seen between the controls and the treated females.
No changes in gravid uterus weight was observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examinations of thyroid
- No changes were seen between the controls and the treated females at macroscopic observations.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examinations of thyroid
- No changes were seen between the controls and the treated females at microscopic observations.

Histopathological findings: neoplastic:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation losses and the mean number of fetuses per dam were not affected by treatment with the test item at all dose levels. In group 3, a statistically significant higher pre-implantation loss was noted. Since pre-implantation loss occurs before treatment start and in the absence of dose-dependency this difference was considered to be incidental.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratios were comparable between the control and the treated groups.

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance was unaffected by treatment.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No treatment related findings were observed.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Observations noted at the skeletal examination were similar between the control and the treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A minimally higher incidence of slight enlargement of brain ventricles (56% of the litters) was noted in high dose foetuses, compared to controls. These changeswere also observed in 32% of control litters, 44% of the low dose litters and 30% of the mid-dose litters. Therefore, the slightly higher incidences in the high dose group are considered to be incidental.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

TAB. 1: SUMMARY OF REPRODUCTION DATA – GROUP DATA

 

Observations	Dose Levels [mg/kg/day]
	0	100	300	1000
No. Dams Inseminated	25	25	25	25
No. Dams That Conceived	25	25	23	25
Percent Dams Conceived	100.0	100.0	92.0	100.0
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	0	0
No. of Litters	25	25	23	25
Total No. Corpora Lutea	276	301	263	303
Mean No. Corpora Lutea/Pregnancy	11.0	12.0	11.4	12.1
Total No. Implantation	269	285	243	293
Mean No. Implants/Pregnancy	10.8	11.4	10.6	11.7
Total No. Live Fetuses	263	281	235	288
Mean No. Live Fetuses/Pregnancy	10.5	11.2	10.2	11.5
Total No. Dead Fetuses	0	0	0	0
Mean No. Dead Fetuses/Pregnancy	0.0	0.0	0.0	0.0
No. Dams with Resorptions and/or Dead Fetuses	6	2	5	4
Total No. Resorptions	6	4	8	5
Mean No. Resorptions/Pregnancy	0.2	0.2	0.3	0.2
Preimplantation Loss (%)(mean)	2.1	4.8	7.6	2.9
Post Implantation Loss (%)(mean)	3.6	1.7	3.3	1.6
Mean Foetal Wt. Live Fetuses	4.13	4.09	4.18	4.11

 

 

TAB. 2: CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE

 

Dose Levels [mg/kg/day]	0		100		300			1000
Observations	a	b	a	b	a	b	a		b
APPEARANCE
Hairloss	1	4.0	0	0.0	1	4.0	2		8.0
Presence of palpable mass	0	0.0	1	4.0	0	0.0	0		0.0
Coloured faeces, yellow	0	0.0	0	0.0	0	0.0	25		100.0

Key:         () = Number of animals alive at start of interval

a = Number of animals affected

b = Percent of animals with observation during interval

 

 

TAB. 3: BODY WEIGHT (g) OF PREGNANT FEMALES - GROUP MEAN DATA

 

Dose Levels [mg/kg/day]	Day of Phase
		0!	3"	6	9	12	15	18	20
0	(n)	25	25	25	25	25	25	25	25
	Mean	215.67	225.12	237.05	244.64	259.30	274.26	306.16	330.42
	SD	11.96	13.19	13.16	13.29	15.88	16.92	21.13	24.61
100	(n)	25	25	25	25	25	25	25	25
	Mean	212.70	220.04	231.39	239.71	252.47	268.15	302.95	327.30
	SD	14.19	17.38	16.13	17.66	17.02	19.15	20.56	25.22
300	(n)	23	23	23	23	23	23	23	23
	Mean	216.02	226.23	236.17	243.73	257.54	274.63	308.58	333.25
	SD	12.82	12.60	12.98	13.41	13.22	18.42	18.59	21.16
1000	(n)	25	25	25	25	25	25	25	25
	Mean	215.65	6	5	4	7	3	6.98	3
	SD	17.29	8.54	9.24	9.35	0.93	2.21	5.26	7.77

 

Note: ! = Gestation phase; " = Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

 

 

TAB. 4: BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES - GROUP MEAN DATA

 

Dose Levels [mg/kg/day]	Day of Phase
		3"	6	9	12	15	18	20
0	(n)	25	25	25	25	25	25	25
	Mean	225.12	237.05	244.64	259.30	274.26	306.16	330.42
	SD	13.19	13.16	13.29	15.88	16.92	21.13	24.61
100	(n)	25	25	25	25	25	25	25
	Mean	220.04	231.39	239.71	252.47	268.15	302.95	327.30
	SD	17.38	16.13	17.66	17.02	19.15	20.56	25.22
300	(n)	23	23	23	23	23	23	23
	M n	226.23	236.17	243.73	257.54	274.63	308.58	333.25
	S	12.60	12.98	13.41	13.22	18.42	18.59	21.16
1000	(n)	25	25	25	25	25	25	25
	Mean	6	5	4	7	3	6.98	3
	SD	8.54	9.24	9.35	0.93	2.21	5.26	7.77

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = mean daily body weight gain over the previous period starting from gestation day 0

 

 

TAB. 5: FOOD CONSUMPTION° (g/animal/day) OF PREGNANT FEMALES – GROUP MEAN DATA

 

Dose Levels [mg/kg/day]	Day of Phase
		3	6	9	12	15	18	20
0	(n)	25	25	25	25	25	25	25
	Mean	8.55	0.90	1.02	2.54	3.82	5.84	6.04
	SD	3.66	2.02	2.67	2.69	1.75	2.60	2.03
100	(n)	25	25	25	25	25	25	25
	Mean	8.55	0.76	0.97	2.09	3.50	6.10	5.81
	SD	3.40	2.51	3.31	2.07	2.24	2.85	1.93
300	(n)	23	23	23	23	23	23	23
	M n	8.56	0.89	1.40	2.77	5.07	7.08	6.41
	S	2.74	2.32	2.27	2.50	3.57	3.15	2.31
1000	(n)	25	25	25	25	25	25	25
	Mean	8.17	0.51	1.25	2.78	3.63	4.88	5.63
	SD	3.58	2.50	2.71	2.84	2.32	2.64	2.75

Note: Data for Dosing/Gestation phase

* = mean value of group is significantly different from control at p < 0.05

** = mean value of group is significantly different from control at p < 0.01

Statistical analysis: Dunnett`s test if group variances are homogeneous

Modified t test if group variances are inhomogeneous ($)

° = food consumed over the previous period starting from Day 0 post coitum

 

 

TAB. 6: TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT, CORRECTED MATERNAL BODY WEIGHT AND CORRECTED MATERNAL BODY WEIGHT GAIN OF FEMALES - GROUP MEAN DATA

 

Dose Levels [mg/kg/day]		Terminal Body weight (g)	Gravid uterus weight (g)	Body weight Day 6 (g)	Corrected maternal body weight^ (g)	Corrected body weight gain# (g)
0	Mean	325.81	64.98	237.96	260.83	23.77
	SD	25.81	15.16	13.15	17.78	8.33
	(n)	25	25	25	25	25
100	Mean	323.78	69.04	231.40	254.74	23.34
	SD	23.03	12.22	16.13	16.84	7.86
	(n)	25	25	25	25	25
300	Mean	329.40	64.38	236.17	265.02	28.84
	SD	21.11	13.17	12.97	18.18	11.89
	(n)	23	23	23	23	23
1000	Mean	327.70	66.29	235.85	261.41	25.56
	SD	26.98	15.89	19.24	32.34	22.56
	(n)	25	25	25	25	25

^ = Corrected maternal body weight at necropsy minus gravid uterus weight

# = Corrected maternal body weight at necropsy minus gravid uterus weight, minus body weight at day 6 of pregnancy

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test

William’s test if group means are different from control at p < 0.05

 

 

TAB. 7: THYROID HORMONE DETERMINATION ON DAY 20 POST COITUM – GROUP MEAN DATA 

 

	Dose Levels [mg/kg/day]	0	100	300	1000
Parameter/units
Triiodothyronine nmol/L	Mean	0.820	0.793	0.880	0.783
	SD	0.134	0.103	0.127	0.113
	n	25	25	25	25
Thyroxine nmol/L	Mean	25.0	23.8	24.7	22.0+
	SD	3.5	3.7	3.7	2.0
	n	25	25	25	25
Thyroid stimulating hormone ng/mL	Mean	7.24	7.51	7.86	7.02
	SD	2.23	2.60	2.49	1.37
	n	25	25	25	25

Controls from group(s): 1 Subgroup(s): 1

* = mean value of group is significantly different from control at p < 0.05

+ = mean value of group is significantly different from control at p < 0.01

 

TAB. 8: LITTER DATA AND SEX RATIOS OF FEMALES - GROUP MEAN DATA

 

Dose Levels [mg/kg/day]		Corpora Lutea	Implan-tations	Uterine Deaths			Viable young			% males	Implantation loss (%)			Litter Weight (g)	Mean Foetal Weight (g)
				Early	Late	Total	Total	Male	Female		Pre	Post	Total		Male	Female	combined
0	Mean	11.04	10.76	0.24	0.00	0.24	10.52	4.60	6.17	45.05	2.11	3.55	5.64	43.31	4.23	4.07	4.13
	SD	2.75	2.57	0.44	0.00	0.44	2.57	1.68	1.83	17.98	4.67	10.18	10.68	11.63	0.73	0.68	0.69
	(n)	25	25	25	25	25	25	25	24	25	25	25	25	25	25	24	25
100	Mean	12.04	11.40	0.16	0.00	0.16	11.24	5.64	5.60	50.64	4.78	1.69	6.40	45.52	4.19	4.00	4.09
	SD	2.26	1.96	0.55	0.00	0.55	2.19	1.96	2.22	15.65	6.96	5.85	8.82	8.87	0.59	0.57	0.56
	(n)	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25
300	Mean	11.44	10.57	0.35	0.00	0.35	10.22	5.30	4.91	51.35	7.60*	3.34	10.91	42.74	4.30	4.07	4.18
	SD	1.70	1.85	0.89	0.00	0.89	2.04	2.20	1.91	17.25	10.45	8.78	11.79	11.02	0.68	0.61	0.62
	(n)	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23	23
1000	Mean	12.12	11.72	0.20	0.00	0.20	11.52	5.96	5.56	51.78	2.94	1.64	4.58	47.35	4.23	4.02	4.14
	SD	1.94	1.84	0.50	0.00	0.50	1.85	1.90	1.98	14.63	7.30	4.13	7.76	8.01	0.59	0.69	0.62
	(n)	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25	25

* = mean value of group is significantly different from control

Statistical analysis: Kruskall Wallis test

William’s test if group means are different from control at p < 0.05

 

 

TAB. 9: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, MALES

 

Parameter/units	Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
ANOGENITAL DISTANCE (mm)	Mean	4.19	4.18	4.13	4.16
	SD	0.37	0.41	0.27	0.32
	(n)	25	25	23	25
Pup weight (g)	Mean	4.23	4.19	4.30	4.23
	SD	0.73	0.59	0.68	0.59
	(n)	25	25	23	25
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓	Mean	2.60	2.60	2.55	2.58
	SD	0.18	0.20	0.20	0.24
	(n)	25	25	23	25

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test

T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

 

TAB. 10: ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA, FEMALES

 

Parameter/units	Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
ANOGENITAL DISTANCE (mm)	Mean	2.50	2.31	2.46	2.48
	SD	0.44	0.43	0.39	0.35
	(n)	24	25	23	25
Pup weight (g)	Mean	4.07	4.00	4.07	4.02
	SD	0.68	0.57	0.61	0.69
	(n)	24	25	23	25
ANOGENITAL DISTANCE ^ (NORMALISED) mm/g⅓	Mean	1.57	1.46	1.55	1.57
	SD	0.25	0.25	0.27	0.24
	(n)	24	25	23	25

^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)

Statistical analysis: Kruskall Wallis test

T test if group mean differences are different from control at p < 0.05

* = mean value of group is significantly different from control

 

 

TAB. 11: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, BRAIN

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	1.828	1.810	1.806	1.810
SD	0.094	0.116	0.109	0.068
Group diff. at p < 0.05		0.067	0.067	0.067
Group diff. at p < 0.01		0.083	0.083	0.083

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 0.26 Df = 3/ 96 F probability = 0.854

Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

TAB. 12: ABSOLUTE ORGAN WEIGHTS (g) - GROUP MEAN DATA, FEMALES, THYROID

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	0.0223	0.0224	0.0244	0.0233
SD	0.0037	0.0041	0.0042	0.0039
Group diff. at p < 0.05		0.0027	0.0027	0.0027
Group diff. at p < 0.01		0.0034	0.0034	0.0034

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.42 Df = 3/ 95 F probability = 0.240

Note: a * indicates group mean is significantly different from control at level of significance shown.

 

 

TAB. 13: ORGAN WEIGHTS° TO BRAIN WEIGHT - GROUP MEAN DATA, THYROID, FEMALES

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Mean	1.220	1.238	1.352	1.288
SD	0.188	0.234	0.261	0.220
Group diff. at p < 0.05		0.153	0.155	0.153
Group diff. at p < 0.01		0.192	0.194	0.192

Data homogeneous by Bartlett's test (Dunnett's test)

Analysis of variance: F ratio = 1.65 Df = 3/ 95 F probability = 0.181

Note: a * indicates group mean is significantly different from control at level of significance shown.

° = expressed as % organ to brain weight ratio

 

 

TAB. 14: MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE - GROUP INCIDENCE, FEMALES

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Whole animal      No abnormalities detected	23	24	22	23
Forelimbs      Hairloss	1	0	1	0
Skin      Subcutaneous mass(es)      Hairloss	0 1	1 0	0 1	0 2
Uterus      Not pregnant      Unilateral implantation	0 1	0 0	2 0	0 0

 

 

TAB. 15: EXTERNAL EXAMINATION OF FOETUSES - GROUP INCIDENCE

 

Group	0 mg/kg/day	100 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
(n)	25	25	25	25
Thyroid THYRO-GLOSSAL DUCT REMNANT	1	1	1	1
Thyroid ECTOPIC THYMIC TISSUE	1	2	0	1

 

 

TAB. 16: MICROSCOPIC OBSERVATIONS – FINAL SACRIFICE - GROUP INCIDENCE

 

Dose Levels [mg/kg/day]	Organ	Cat	Observations	No. Foetuses			No. Litters
				Observed	Affected	%	Observed	Affected	%
0	Forelimb	AN	Short	263	1	0.38	25	1	4.00
	Head	AN	Domed shape	263	1	0.38	25	1	4.00
	Hindlimb	AN	Short	263	1	0.38	25	1	4.00
	Mouth	AN	Abnormal shape	263	1	0.38	25	1	4.00
	Whole Foetus		No abnormalities detected	263	262	99.62	25	24	96.00
100	Whole Foetus		No abnormalities detected	281	281	100.00	25	25	100.00
300	Tail	AN	Short	235	1	0.43	23	1	4.35
	Whole Foetus		No abnormalities detected	235	234	99.57	23	23	100.00
1000	Hindlimb	AN	Abnormal shape	288	2	0.69	25	2	8.00
	Whole Foetus		No abnormalities detected	288	286	99.31	25	25	100.00

 

 

TAB. 17: FIXED VISCERAL EXAMINATION OF FOETUSES - GROUP INCIDENCE

 

Dose Levels [mg/kg/day]	Organ	Cat	Observation(s)	No. Foetuses			No. Litters
				Obs	Aff	%	Obs	Aff	%
0	Abdomen	VA	Umbilical vein left	126	11	8.73	25	8	32.00
	Abdomen	VA	Haemorrhagic	126	3	2.38	25	3	12.00
	Brain	AN	Ventricles enlarged moderate	126	1	0.79	25	1	4.00
	Brain	VA	Ventricles enlarged slight	126	12	9.52	25	8	32.00
	Ear	MA	Malpositioned	126	1	0.79	25	1	4.00
	Forelimb	MA	Focomelia	126	1	0.79	25	1	4.00
	Head	MA	Ankyloglossia	126	1	0.79	25	1	4.00
	Heart	AN	Septum abnormal size thin	126	1	0.79	25	1	4.00
	Hindlimb	MA	Focomelia	126	1	0.79	25	1	4.00
	Kidneys	VA	Pelvic dilatation slight	126	9	7.14	25	5	20.00
	Mouth	MA	Nasal conchae absent	126	1	0.79	25	1	4.00
	Tail	AN	Bent	126	1	0.79	25	1	4.00
	Testis	AN	Displaced	126	1	0.79	25	1	4.00
	Ureter	AN	Enlarged moderate	126	1	0.79	25	1	4.00
	Ureter	VA	Enlarged slight	126	5	3.97	25	3	12.00
100	Abdomen	VA	Umbilical vein left	134	8	5.97	25	5	20.00
	Abdomen	VA	Haemorrhagic	134	2	1.49	25	2	8.00
	Brain	AN	Ventricles enlarged moderate	134	1	0.75	25	1	4.00
	Brain	VA	Ventricles enlarged slight	134	20	14.93	25	11	44.00
	Great vessels	VA	Innominate artery longer	134	1	0.75	25	1	4.00
	Kidneys	VA	Pelvic dilatation slight	134	11	8.21	25	7	28.00
	Testis	AN	Displaced	134	4	2.99	25	3	12.00
	Ureter	VA	Enlarged slight	134	7	5.22	25	4	16.00
300	Abdomen	VA	Umbilical vein left	111	5	4.50	23	5	21.74
	Brain	VA	Ventricles enlarged slight	111	10	9.01	23	7	30.43
	Great vessels	VA	Innominate artery longer	111	2	1.80	23	2	8.70
	Heart	AN	Septum abnormal size thin	111	1	0.90	23	1	4.35
	Kidneys	VA	Pelvic dilatation slight	111	8	7.21	23	6	26.09
	Testis	AN	Displaced	111	1	0.90	23	1	4.35
	Ureter	VA	Enlarged slight	111	4	3.60	23	4	17.39
1000	Abdomen	VA	Haemorrhagic	138	1	0.72	25	1	4.00
	Abdomen	VA	Umbilical vein left	138	13	9.42	25	11	44.00
	Brain	AN	Ventricles enlarged moderate	138	1	0.72	25	1	4.00
	Brain	VA	Ventricles enlarged slight	138	23	16.67	25	14	56.00
	Great vessels	VA	Innominate artery longer	138	1	0.72	25	1	4.00
	Heart	AN	Septum abnormal size thin	138	1	0.72	25	1	4.00
	Kidneys	AN	Pelvic dilatation moderate	138	1	0.72	25	1	4.00
	Kidneys	VA	Pelvic dilatation slight	138	9	6.52	25	5	20.00
	Testis	AN	Displaced	138	5	3.62	25	5	20.00
	Thoracic cavity	AN	Haemorrhage	138	1	0.72	25	1	4.00
	Ureter	AN	Enlarged moderate	138	1	0.72	25	1	4.00
	Ureter	VA	Enlarged slight	138	8	5.80	25	5	20.00

 

 

TAB. 18: SKELETAL EXAMINATION OF FOETUSES - GROUP INCIDENCE 

 

Dose Levels [mg/kg/day]	Organ	Cat	Observation(s)	No. Foetuses			No. Litters
				Obs	Aff	%	Obs	Aff	%
0	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	137	17	12.41	24	7	29.17
	Ribs	AN	Wavy	137	13	9.49	24	8	33.33
	Ribs	VA	14 ribs	137	16	11.68	24	8	33.33
	Ribs	VA	Short 14th	137	60	43.80	24	21	87.50
	Ribs	VA	Rudimentary 14th	137	17	12.41	24	13	54.17
	Skull	AN	Temporal incomplete ossification	137	10	7.30	24	7	29.17
	Skull	VA	Parietal incomplete ossification	137	24	17.52	24	12	50.00
	Skull	VA	Interparietal incomplete ossification	137	47	34.31	24	17	70.83
	Skull	VA	Hyoid incomplete ossification	137	2	1.46	24	2	8.33
	Skull	VA	Supraoccipital incomplete ossification	137	47	34.31	24	18	75.00
	Sternebrae	AN	Asymmetrical ossification	137	3	2.19	24	3	12.50
	Sternebrae	AN	Asymmetrical ossification 5th	137	4	2.92	24	4	16.67
	Sternebrae	VA	Incomplete ossification 6th	137	17	12.41	24	9	37.50
	Sternebrae	VA	No ossification 5th	137	3	2.19	24	2	8.33
	Sternebrae	VA	Incomplete ossification 5th	137	13	9.49	24	12	50.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	137	2	1.46	24	2	8.33
100	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	147	7	4.76	25	6	24.00
	Ribs	AN	Wavy	147	6	4.08	25	4	16.00
	Ribs	VA	Short 14th	147	62	42.18	25	22	88.00
	Ribs	VA	Rudimentary 14th	147	19	12.93	25	14	56.00
	Ribs	VA	14 ribs	147	12	8.16	25	8	32.00
	Skull	AN	Temporal incomplete ossification	147	8	5.44	25	6	24.00
	Skull	VA	Supraoccipital incomplete ossification	147	50	34.01	25	15	60.00
	Skull	VA	Parietal incomplete ossification	147	18	12.24	25	10	40.00
	Skull	VA	Interparietal incomplete ossification	147	35	23.81	25	14	56.00
	Skull	VA	Hyoid incomplete ossification	147	5	3.40	25	4	16.00
	Sternebrae	AN	Asymmetrical ossification	147	2	1.36	25	2	8.00
	Sternebrae	AN	Asymmetrical ossification 5th	147	7	4.76	25	5	20.00
	Sternebrae	AN	Bipartite 5th	147	2	1.36	25	1	4.00
	Sternebrae	VA	Incomplete ossification 6th	147	5	3.40	25	4	16.00
	Sternebrae	VA	Incomplete ossification 5th	147	10	6.80	25	7	28.00
	Sternebrae	VA	No ossification 5th	147	3	2.04	25	2	8.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	147	3	2.04	25	2	8.00
300	Cervical vertebrae	AN	Cervical rib(s)	124	1	0.81	23	1	4.35
	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	124	7	5.65	23	5	21.74
	Ribs	AN	Wavy	124	11	8.87	23	7	30.43
	Ribs	VA	Short 14th	124	48	38.71	23	21	91.30
	Ribs	VA	14 ribs	124	8	6.45	23	5	21.74
	Ribs	VA	Rudimentary 14th	124	17	13.71	23	12	52.17
	Skull	AN	Temporal incomplete ossification	124	6	4.84	23	4	17.39
	Skull	VA	Supraoccipital incomplete ossification	124	35	28.23	23	12	52.17
	Skull	VA	Interparietal incomplete ossification	124	23	18.55	23	12	52.17
	Skull	VA	Parietal incomplete ossification	124	17	13.71	23	9	39.13
	Sternebrae	AN	Asymmetrical ossification 5th	124	3	2.42	23	3	13.04
	Sternebrae	AN	Asymmetrical ossification	124	2	1.61	23	2	8.70
	Sternebrae	VA	Incomplete ossification 6th	124	10	8.06	23	5	21.74
	Sternebrae	VA	Incomplete ossification 5th	124	11	8.87	23	8	34.78
	Thoracic vertebrae	VA	Centrum incomplete ossification	124	6	4.84	23	6	26.09
1000	Forepaw(s)	AN	Metacarpal(s) no ossification 4th	150	5	3.33	25	4	16.00
	Ribs	AN	Wavy	150	10	6.67	25	6	24.00
	Ribs	VA	Rudimentary 14th	150	19	12.67	25	15	60.00
	Ribs	VA	Short 14th	150	44	29.33	25	21	84.00
	Ribs	VA	14 ribs	150	15	10.00	25	7	28.00
	Sacral vertebrae	AN	Arch(es) incomplete ossification	150	1	0.67	25	1	4.00
	Skull	AN	Temporal incomplete ossification	150	9	6.00	25	4	16.00
	Skull	AN	General incomplete ossification	150	5	3.33	25	2	8.00
	Skull	VA	Interparietal incomplete ossification	150	28	18.67	25	16	64.00
	Skull	VA	Supraoccipital incomplete ossification	150	44	29.33	25	17	68.00
	Skull	VA	Parietal incomplete ossification	150	14	9.33	25	10	40.00
	Skull	VA	Hyoid incomplete ossification	150	3	2.00	25	3	12.00
	Sternebrae	AN	Asymmetrical ossification	150	3	2.00	25	3	12.00
	Sternebrae	AN	Asymmetrical ossification 5th	150	4	2.67	25	3	12.00
	Sternebrae	VA	Incomplete ossification 5th	150	8	5.33	25	6	24.00
	Sternebrae	VA	Incomplete ossification 6th	150	7	4.67	25	5	20.00
	Sternebrae	VA	No ossification 5th	150	1	0.67	25	1	4.00
	Thoracic vertebrae	VA	Centrum asymmetrical dumb-bell shaped	150	1	0.67	25	1	4.00
	Thoracic vertebrae	VA	Centrum incomplete ossification	150	1	0.67	25	1	4.00

 

Applicant's summary and conclusion

Conclusions:

On the basis of the results, the dosage of 1000 mg/kg/day is considered the NOAEL for maternal and embryo-foetal development.

Executive summary:

In a GLP-conform study according to OECD guideline 414, the effects of the test item on pregnant female Wistar Han rats and embryo-fetal development were assessed when administered orally by gavage once daily to mated female rats from Day 6 through to Day 19 post coitum, inclusive. Each group consisted of 25 mated female rats. Each group consisted of 25 mated female rats. The test material was administered once daily at dose levels of: 0 mg/kg bw/day (vehicle control); 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight  was used. Control animals were dosed with the vehicle alone (0.5 % carboxy- methylcellulose (CMC)).

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Thyroid hormone determination was performed. The brain and thyroid were weighed. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, gravid uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No mortality occurred during the study. All females were pregnant with the exception of 2 females in the mid- dose group. Unilateral implantation was observed in one control female. The number of dams with live foetuses were 25 each in the control, low and high dose groups and 23 in the mid dose group. No treatment related clinical signs were described. Yellow faeces were noted for all high dose females during the last days of gestation period. Maternal body weight and body weight gain were unaffected by treatment. Food consumption was comparable between groups. No changes in thyroid hormone levels were observed. No changes in terminal body weight, gravid uterus weight or absolute weight gain was observed. No changes in organ weights were seen between the controls and the treated females. Litter data and sex rations was comparable between the control and the treated groups. Anogenital distance was unaffected by treatment. No changes were seen between the controls and the treated females at macroscopic or microscopic observations. Regarding the external, visceral and skeletal examinations of foetuses, no treatment related abnormal findings were observed.