Trisodium, bis [2-((E)-(4-hydroxy-2-oxido-3-((E)-(5-sulfonatonaphthalen-1-yl)diazenyl)phenyl)diazenyl)benzoate] chromate(3-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 13 July 2017 to 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 17. May. to 03. Jun. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

The purpose of this study is to investigate the effects of the test item on pregnancy and embryo-foetal development in the rat, following oral administration in order to select the dosages for a main study.
The required amount of test item will be dissolved/suspended in vehicle. The formulation will be prepared daily (concentrations of 5, 50 and 100 mg/mL), unless specified otherwise. Concentrations will be calculated and expressed in terms of test item as supplied.

GLP compliance:

no

Remarks:

carried out in a GLP compliant facility

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo Holland, Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females: nulliparous and non-pregnant until mating phase
- Age at study initiation: females: 11 to 12 weeks; males: at least 11 weeks
- Weight at study initiation: females: 200 to 225 g; males: at least 340 g
- Fasting period before study:
- Housing: pre-pairing period: up to 5 animals of one sex only per cage, in polysulfone cages measuring 59.5 x 38 x 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy), nesting material (Scobis 0 Mucedola) provided inside suitable bedding bags as necessary and changed at least 3 times a week
- Housing: mating period: 1 male to 1 female in clear polysulfone cages measuring 42.5 x 26.6 x 18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese, Italy), each try held absorbent material which was inspected and changed daily
- Diet: ad libitum; commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G Galilei, 4, 20019 Settimo Milanese MI, Italy); analyses kept on file at testing facility
- Water: ad libitum; via water bottles; analyses kept on file at testing facility
- Acclimation period: 20 days during which a health evaluation was made
- Identification: tattoo on the hind feet

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The required amount of test item was dissolved/suspended in vehicle to make 10 ml/kg bw doses. The formulations were prepared daily, and the concentrations were calculated and expressed in terms of test item as supplied.

Details on mating procedure:

- Impregnation procedure: cohousing
- M/F ratio per cage: males were housed individually; 1 female was paired to each male home cage and left overnight
- Length of cohabitation: until mating confirmed
- Further matings after two unsuccessful attempts: animals were cohoused until a positive identification of mating was made
- Proof of pregnancy: presence of sperm on the vaginal smear or presence of a copulating plug was considered the day of mating; referred to as Day 0 of gestation (Day 0 post coitum)
- Other: full mating records were maintained

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

It is the responsibility of the Sponsor to determine the identity, strength, purity, composition, stability and other characteristics of the test item for each batch of test item before its use in the study. A certificate of analysis for the test item should also be supplied.

Duration of treatment / exposure:

All females were administered a single daily oral dose

Frequency of treatment:

Daily

Details on study schedule:

From Day 6 to Day 19 post coitum

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

low-dose group

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

mid-dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high-dose group

No. of animals per sex per dose:

6 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Housing post coitum: 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. Each female was identified within the study by ear notch and housed no more than 2 to a cage. The cages were identified by a label recording the study number, animals numbers and details of the treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects. Each dose group comprised 6 mated female rats.

Positive control:

none

Parental animals: Observations and examinations:

MORTALITY / VIABILITY: yes
- Time schedule: daily: early in the working day and then again in the afternoon

CLINICAL SIGNS: yes
- Time schedule: daily

CAGE TRAY: yes
- Time schedule: daily, from day 3 (not tabulated)

BODY WEIGHT: yes
- Time schedule: days 0, 3, 6, 9, 12, 15, 18 and 20 (terminal) post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: no

WATER CONSUMPTION AND COMPOUND INTAKE: no

Litter observations:

STANDARDISATION OF LITTERS : no

PARAMETERS EXAMINED
The following parameters were examined in offspring:
- External examinations: yes; all litter
- Number, sex and weight of all live foetuses
- Number and sex of dead foetuses
- Anomalies (small foetal weight = anomalies)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: all surviving animals were sacrificed on Day 20 post coitum; by carbon dioxide.

GROSS NECROPSY
- Macroscopic observations: females were examined visually externally.

ORGAN WEIGHTS
- Organs examined: ovaries and uterine content.

Postmortem examinations (offspring):

SACRIFICE
- All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia on Day 20 post coitum

GROSS NECROPSY
- Gross necropsy consisted of external visual examination
- Findings of small foetus was classified as anomalies

HISTOPATHOLOGY / ORGAN WEIGTHS: no

SYMPTOMS
- Malformations: major abnormalities that are rare and/or affect the survival or health of the species under investigation. Foetuses with apparent malformations will be retained in Bouin fixative.
- 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 was assessed by Dunnett's test or a modified t test, depending on nthe homogeneity of data. Statistical analysis of non-continuous variables was 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.

Reproductive indices:

Litter data:
Pre-implantation loss was calculated as a percentage from the following:
Pre-implantation loss % = (number of corpora lutea - number of implantations) / number of corpora lutea x 100
Post-implantation loss was calculated as a percentage from the following:
Post-implantation loss % = (number of implantations - number of live foetuses) / number of implantations x 100
Total implantation loss was calculated as a percentage from the formula:
Total implantation loss % = (number of corpora lutea - number of live foetuses) / number of corpora lutea x 100
Sex ratios of the foetuses were calculated as the percentage of males per litter.
All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group. as well as in terms of the mean litter percentage of affected litters.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No signs of reactions to treatment were noted during the dosing period, however one high-dose female demonstrated piloerection (days 9-12) and teeth missing (days 12 to 15), which was considered to be incidental and not treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight were noted between control and treated groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Red faeces (slight) were observed in cage tray observations of the mid- and high-dose groups, generally starting from 3 to 4 days after the start of treatment. This finding was due to the colour of the test item and its properties as a dye.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Description (incidence and severity):

Litter data, mean foetal weight and sex ratios were not affected by treatment.
External examination of the foetuses demonstrated no abnormalities, with the exception of one incidence of small foetus by weight in the mid-dose group which was considered to be incidental.

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Reproductive effects observed:

no

Table 1: Fate of females - group incidence.

Group	1	2	3	4
Initial group size	6	6	6	6
Unilateral implantation	1	0	0	0
Unilateral total resorption	0	1	0	0
Not pregnant	0	0	1	0
With live foetuses at gestation Day 20	6	5	5	6

Table 2: Clinical signs of females - group incidence between days 0 and 20.

Group:	1		2		3		4
Number in group:	6		6		6		6
	no.	%	no.	%	no.	%	no.	%
Appearance
Piloerection	0	0	0	0	0	0	1	16.7
Eye, ear, mouth
Tooth/teeth missing	0	0	0	0	0	0	1	16.7

Table 3: Mean body weight (g) of pregnant females.

Group		Day of phase
		0 (gestation)	3 (dosing/ gestation)	6	9	12	15	18	20
1	no.	6	6	6	6	6	6	6	6
	mean	224.80	237.28	246.72	257.48	268.10	283.23	318.72	347.44
	s.d.	4.27	4.52	3.15	4.82	5.37	6.15	7.23	11.34
2	no.	5	5	5	5	5	5	5	5
	mean	231.15	239.73	249.67	261.20	271.27	287.28	324.55	357.27
	s.d.	9.38	9.14	6.66	6.17	5.95	7.61	11.79	17.16
3	no.	5	5	5	5	5	5	5	5
	mean	224.99	238.25	249.22	259.32	270.20	288.11	321.36	356.76
	s.d.	7.46	9.21	10.09	8.71	12.36	8.01	19.03	18.02
4	no.	6	6	6	6	6	6	6	6
	mean	229.02	243.78	252.51	256.60	269.74	284.67	318.38	344.04
	s.d.	6.58	7.56	8.36	11.72	10.35	8.87	15.17	20.77

Table 4: Mean body weight (g) gain per day starting from day 0 of pregnant females.

Group		Day of phase
		3	6	9	12	15	18	20
1	no.	6	6	6	6	6	6	6
	mean	4.159	3.149	3.585	3.539	5.044	11.829	14.362
	s.d.	1.129	0.990	1.414	1.032	1.327	1.706	2.150
2	no.	5	5	5	5	5	5	5
	mean	2.861	3.315	3.841	3.358	5.335	12.423	16.360
	s.d.	1.501	1.329	1.591	2.074	1.713	2.854	2.973
3	no.	5	5	5	5	5	5	5
	mean	4.419	3.657	3.366	3.626	5.972	11.084	17.697
	s.d.	1.485	0.482	1.040	1.852	2.080	4.499	1.672
4	no.	6	6	6	6	6	6	6
	mean	4.918	2.912	1.361	4.380	4.979	11.234	12.833
	s.d.	0.952	0.820	4.773	1.082	1.129	3.299	3.479

Table 5: Mean terminal body weight, gravid uterus weight and absolute weight gain (g) of females.

Group		Terminal body weight (g)	Gravid uterus weight (g)	Absolute weight gain (g)
1	no.	6	6	6
	mean	342.68	66.45	51.47
	s.d.	10.15	10.28	4.86
2	no.	5	5	5
	mean	352.10	73.54	47.42
	s.d.	19.35	18.00	8.14
3	no.	5	5	5
	mean	352.98	76.02	52.00
	s.d.	20.64	12.19	8.45
4	no.	6	6	6
	mean	338.92	65.15	44.77
	s.d.	22.14	20.02	7.86

Table 6: Mean litter data and sex ratios of females.

Group		Corpora lutea	Implantations	Uterine deaths: early	Uterine deaths: late	Uterine deaths: total	Viable young: M	Viable young: F	Viable young: total	% males	Implantation loss: pre (%)	Implantation loss: post (%)	Implantation loss: total (%)	Litter weight (g)	Mean foetal weight (g)
1	no.	6	6	6	6	6	6	6	6	6	6	6	6	6	6
	mean	12.67	12.67	0.67	0.00	0.67	5.83	6.17	12.00	47.85	0.00	4.85	4.86	44.13	3.69
	s.d.	2.50	2.50	0.82	0.00	0.82	2.14	1.17	2.19	10.91	0.00	5.53	5.54	7.81	0.22
2	no.	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	mean	13.60	13.40	0.80	0.00	0.80	7.00	5.60	12.60	56.51	1.54	6.34	7.49	48.36	3.83
	s.d.	2.61	2.70	1.30	0.00	1.30	1.58	2.51	3.21	9.14	3.44	10.83	13.33	12.70	0.20
3	no.	5	5	5	5	5	5	5	5	5	5	5	5	5	5
	mean	14.00	14.00	0.40	0.00	0.40	8.60	5.00	13.60	63.04	0.00	3.24	3.25	48.16	3.57
	s.d.	2.24	2.24	0.55	0.00	0.55	2.51	1.58	2.61	10.12	0.00	4.49	4.50	7.06	0.24
4	no.	6	6	6	6	6	6	6	6	6	6	6	6	6	6
	mean	13.33	13.17	1.83	0.00	1.83	6.17	5.17	11.33	50.99	1.12	13.02	14.13	42.07	3.65
	s.d.	1.51	1.33	4.02	0.00	4.02	3.06	1.94	3.72	17.58	2.74	28.73	28.26	15.03	0.31

Table 7: Group incidence of macroscopic observations of females.

Group:	1	2	3	4
Number in group:	6	6	6	6
Uterus
Not pregnant:	0	0	1	0
Unilateral implantation:	1	0	0	0
Unilateral total resorption:	0	1	0	0
Whole animal
No abnormalities detected:	5	5	5	6

Table 8: External examination of foetuses - group incidence.

Group	Organ	Cat	Obsertation(s)	No. foetuses			No. litters
				observed	affected	%	observed	affected	%
1	whole foetus		no abnormalities detected	72	72	100.00	-	-	-
2	whole foetus		no abnormalities detected	63	63	100.00	-	-	-
3	whole foetus		no abnormalities detected	68	67	98.53	-	-	-
	whole foetus	AN	small foetus	68	1	1.47	5	1	20.00
4	whole foetus		no abnormalities detected	68	68	100.00	-	-	-

Conclusions:

Up to 1000 mg/kg bw/day test item did not induce toxic effects in dams or foetuses.

Executive summary:

Toxicological effects of the test item during pregnancy and embryo-foetal development were evaluated in an experimental study. Female Sprague-Dawley rats were mated then administered 50, 500 or 1000 mg/kg bw test item (6 animals per dose group) in softened water by oral gavage daily on days 6 to 19 post coitum. A negative control group was simultaneously administered softened water only.

No clinical signs nor significant signs of reaction to treatment were noted in treated females. No differences were noted in body weight, gravid uterus weight, litter data or macroscopic observation of treated females, when compared to controls. On the basis of the above results, it can be concluded that the test item did not induce any toxic effects in dams or foetuses up to and including the high dose tested of 1000 mg/kg day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-320-4

EC Name:

-

Cas Number:

94933-05-8

Molecular formula:

not applicable

IUPAC Name:

chromium(3+) trisodium bis(2-(2-{4-hydroxy-2-oxido-3-[2-(5-sulfonatonaphthalen-1-yl)diazen-1-yl]phenyl}diazen-1-yl)benzoate)

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS s.r.l., San Pietro al Natisone (UD), Italy
- Females nulliparous and non-pregnant: yes, virgin
- Age: males, females: 7-8 weeks on arrival
- Weight at study initiation: males 200-225 g; females 175-200 g on arrival
- Housing: from arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
- Diet: a commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l.) was offered ad libitum throughout the study.
- Water: Drinking water was supplied ad libitum to each cage via water bottles.
(There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking tap water or the diet)
- Acclimation period: 29 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
-Allocation to groups: on the day of allocation (7 days prior to the start of treatment), all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage. During the pre-test period, two allocated females showing irregular cycle were substituted with stock females checked for regular cycles. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were evenly distributed across the battery to minimise possible environmental effects.
Females were selected on the basis of pre-exposure oestrous cyclicity and animals that failed to exhibit regular cycles were not included in the groups. Oestrous cycle was monitored by vaginal smears in all stock females for at least 1 week before allocation, in order to exclude from the study females with irregular cycle.


ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 15 %
- Air changes: 15 to 20 air changes per hour
- Photoperiod: artificial light for 12 hours each day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

FORMULATION PROCEDURE
The required amount of test item was dissolved in the vehicle. The formulationswere prepared weekly (concentrations of 5, 20 and 80 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

Details on mating procedure:

Mating was monogamous (one male to one female) with the exception indicated below. A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred. Female animal no. X0690075 was excluded from the group mean calculation of the body weight and body weight gain at the start of the mating phase, since it was assigned to the phase one day later respect to the other females due to the mortality observed in male animal no. X0690076.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the formulation procedure and to verify the stability of the formulations.
The analytical method was validated in the range from 1 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in SOPs for solutions (r > 0.98; accuracy 90-110 %; precision CV <5 %). In the same study, a 28 hour stability at room temperature and an 8 day stability at +5 ºC ± 3 ºC were verified in the range from 1 to 100 mg/mL. The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration) to confirmthat the method was suitable. All results for all levels were within the acceptability limits stated in SOPs for concentration (90-110 %).
Samples of the formulations prepared on two occasions during the study (Week 1 and again towards the end of the study) were analysed to check the concentration.
Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was SkanIt software version 2.4.2.55. Results of the analyses were within the acceptability limits stated in SOPs for concentration of solutions (90-110 %).

Duration of treatment / exposure:

-Males: 28 or 29 days (for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Days 29 and 30))
-Females: at least 51 days (for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum.
Four not pregnant females were dosed up to the day before necropsy.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7and 13 post partum.

Frequency of treatment:

once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight (adjusted once per week for each animal according to the last recorded body weight)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight (adjusted once per week for each animal according to the last recorded body weight)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight (adjusted once per week for each animal according to the last recorded body weight)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day

Remarks:

administered at a constant volume of 10 ml/kg body weight (adjusted once per week for each animal according to the last recorded body weight)

No. of animals per sex per dose:

10 males and 10 females for each dose level and control

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected by the Sponsor, based on information from a preliminary non-GLP compliant study.

Examinations

Parental animals: Observations and examinations:

MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.

CLINICAL SIGNS
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.
Observations of the cage tray during the pre-mating period were performed for all groups and were recorded three times weekly.
During pairing period, after mating (males) and during gestation/post partum periods (females) observations of the cage tray were performed and were recorded daily for all groups.

BODY WEIGHT
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

PARTURITION CHECK AND DURATION OF GESTATION
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. X0690035 and X0690037 (Group 2), X0690047 (Group 3) and X0690073 (Group 4) which did not give birth after 25 days of post coitum period were sacrificed shortly after.
These animals were found not pregnant or showed unilateral total resorption at necropsy. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth when the parturition was defined complete (Day 0 post partum).

BLOOD COLLECTION
As a part of the necropsy procedure, approximately 0.8 mLof blood samples were withdrawn under isofluorane anaesthesia from the abdominal vena cava from all parental male and female rats.
All blood samples mentioned above were transferred into tubes of appropriate capacity, containing no anticoagulant and centrifuged at roomtemperature. The serumobtained was divided in several aliquots for analysis. The aliquots obtained were stored at -80 °C until analysis.

BIOANALYSIS - THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K). Samples from all parental males from all groups
The results of these analyses are presented as individual data, mean and standard deviation and reported in ng/mL. Since no treatment related effects were seen in the determination performed in parental males, samples obtained from females were not analysed.
When the serum levels for Total triiodothyronine (total T3) were BLOQ (below the limit of quantification - 3.75 ng/mL), the values were not included in the mean calculation.

Oestrous cyclicity (parental animals):

Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identification of mating including 2 weeks before the pairing. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy.

Litter observations:

PUPS IDENTIFICATION, WEIGHT AND OBSERVATION
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. The data were reported for Days 1, 4 and 13 post partum. The data of Day 7 post partum are not tabulated in this report but will be archived together with all other raw data.
Pups dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

ADJUSTMENT OF LITTER SIZE (CULLING) AND PUPS SELECTION FOR BLOOD COLLECTION AT NECROPSY
On Day 4 post partum, all pups were weighed and litters in excess of 8 offspring were culled to 8 (4 males and 4 females, where possible) by a random selection. At least two pups (males or females, selected for culling) were sacrificed for blood collection. Two male pups were selected for dam nos. X0690053 and X0690057 (Group 3) and two female pups were selected for dam nos. X0690039 (Group 2), X0690045 (Group 3) and X0690075 (Group 4).
For litters with 8 or less pups, two female pups were sacrificed for blood collection for dam nos. X0690033 (Group 2) and X0690065 (Group 4). One female pup was selected for dam nos. X0690043 (Group 3) and X0690067 (Group 4). No culling was performed for dam no. X0690027 (Group 2) due to a reduced size of the litter.

NIPPLE COUNT AT DAY 13 POST PARTUM
No nipples/areolae in male pups were present.

ANOGENITAL DISTANCE (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.

BLOOD COLLECTION FOR THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
On Day 4 post partum, blood samples (approximately 0.2 mL) were collected from two of the culled pups (males or females, where possible). Blood samples were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture).
On Day 14 post partum, blood samples (approximately 0.5 mL) were withdrawn under light ether anaesthesia from the heart (by intracardiac puncture) from at least two pups (1 sample/sex, if possible) per litter.

BIOANALYSIS - THYROID HORMONE DETERMINATION (T3, T4 AND TSH)
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by a multiplex assay, using LuminexMagpix system and the MILLIPLEX MAP Rat ThyroidMagnetic Bead Panel kit (MerkMillipore, cat. no. RTHYMAG-30K).
Samples from pups on Day 14 post partum. The results of these analyses are presented as individual data, mean and standard deviation and reported in ng/mL.
Since no treatment related effects were seen in the determination performed in pups on Day 14 post partum, samples obtained from pups on Day 4 post partum were not analysed.
When the serum levels for Total triiodothyronine (total T3) were BLOQ (below the limit of quantification - 3.75 ng/mL), the values were not included in the mean calculation.

Postmortem examinations (parental animals):

Parental animals were killed by exsanguination under isofluorane anaesthesia. Surviving males were killed after the mating of all females on Days 29 and 30 of the study. The females with live pups were killed on Day 14 post partum. The non pregnant females were killed after Day 25 post coitum.

NECROSCOPY AND BLOOD COLLECTION
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (with the exception of the organs from the animal found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of apparently non-pregnant female or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
From all animals completing the scheduled test period, the organs indicated in "any other information on materials and methods" were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

TISSUES FIXED AND PRESERVED
Samples of all the tissues (all parental animals) listed in "any other information on materials and methods" were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGICAL EXAMINATION
The tissues required for histopathological examination are listed in "any other information on materials and methods", After dehydration and embedding in paraffin wax, sections of the tissueswere cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
i) Tissues specified in "any other information on materials and methods" from all males and all females in the control and high dose group killed at term.
ii) Tissues specified in "any other information on materials and methods" from the animal dying during the treatment period (no. X0690076).
iii) All abnormalities in all groups.
A detailed qualitative evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject: Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS- H stained sections were used to identify the spermatogenic stages.

Postmortem examinations (offspring):

Pups that had completed the scheduled test period (Day 4 post partum or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
Pups selected for blood collection for hormone determination were killed on the day of blood sampling.

NECROPSY AND BLOOD COLLECTION
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

NIPPLE COUNT RETENTION AT DAY 14 POST PARTUM
The ventral region of male pups was checked for presence of nipples/areolae.


ORGAN WEIGHTS
Thyroids were weighed from one male and one female pups selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin.
The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

see "any other information on materials and methods"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In male animals receiving 800 mg/kg bw/day, red/brown staining on the tail was observed, generally starting at the end of pre-mating period up until the end of observation period.
No clinical signs were observed in males of the low and mid-dose groups. In female animals, red/brown staining on the tail was generally observed starting from the second week of the pre-mating period up until the end of the post partum period in high dose females and only during the gestation and post partum period in mid-dose females. In addition, hairloss on the lower forelimb was observed in one high dose female (no. X0690073).
Observations of the cage tray
– Red staining (slight to marked) and red faeces on the cage tray were observed during the pre-mating and mating periods in the animals of high dose group of both sexes (800 mg/kg/day). For males, the signs were observed also after mating up to the end of the treatment.
– Red faeces on the cage tray were observed during the mating period in the animals of mid-dose group (200 mg/kg/day). This sign was present in males also after mating up to the end of treatment.
– Red staining (slight) and red faeces on the cage tray were generally seen during the gestation and post partum periods in females receiving dose levels of 200 and 800mg/kg/day.
The above mentioned findings were considered related to the colour of the test item which was eliminated by the animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male receiving 800 mg/kg/day was found dead on Day 7 of the study (no. X0690076, during the pre-mating phase). No clinical signs were observed in the animal prior to death.
Macroscopically, the most relevant findings observed in this animal included dark fluid contents in the thoracic cavity and dark colour of lungs. Microscopically, the most relevant findings were chronic inflammation and necrosis in the lungs with brown foreign material (test item like) and brown pigment-laden macrophages in the alveoli of the lungs, associated with myocardial necrosis in the heart and congestion/haemorrhage of kidneys.
On the basis of these findings, the cause of death was attributed to a misdosing. All other gross pathological findings were considered incidental.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption in treated males and females was comparable to the control group during the study.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At microscopic observations, performed in the animals sacrificed at the end of the study, no treatment-related changes were noted. The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle were similar between treated and control animals.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

SPERMATOGENIC CYCLE
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-coital intervals and copulatory index were similar between treated and control animals.
Fertility index both for males and females was 100 % each in the control and Group 4, while 80 % in Group 2 and 90 % in Group 3. Two males in Group 2 (nos. X0690036 and X0690038) and one in Group 3 (no. X0690048) failed to induce pregnancy. These isolated cases were considered incidental.
Two females (nos. X0690035 and X0690037) receiving 50 mg/kg/day and one female (no. X0690047) receiving 200 mg/kg/day were proved not pregnant at necropsy. One female receiving 800 mg/kg/day (no. X0690073) had unilateral total resorption in the left horn and was not pregnant in the right one.
The number of females with live pups on Day 14 post partum was: 10 in the control, 8 in the low dose (50 mg/kg/day), 9 in the mid-dose (200 mg/kg/day) and 9 in the high dose group (800 mg/kg/day).

Details on results (P0)

THYROID HORMONE DETERMINATION
No changes in thyroid hormones were recorded in parental male animals of all treated groups.

IMPLANTATION, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES
Gestation periods were similar between treated females and control group. All pregnant dams gave birth on Day 22 post coitum (mean value).
Numbers of corpora lutea, implantation sites and pre-implantation loss of treated females were comparable to the control group. Pre-natal loss (expressed as percentage) was slightly higher in treated groups, when compared to the control values. This difference was not significant at statistical analysis, and without dose relationship, and thus considered incidental and of no toxicological relevance.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Apparently no food intake (milk), small appearance, cold to touch and pale aspect were the main clinical signs noted in control and treated pups. One pup at the low dose level (no.1; 50 mg/kg/day) showed first black tail (first days after birth), and later tip of tail missing (from the second week of age). In addition this pup showed also swollen/damaged left hindlimb and hairloss on the sacral region. Tip of tail missing was also noted in one pup (no. 14) of the mid-dose group (200 mg/kg/day). In addition, hairloss on the head was noted in all pups of dam no. X0690043 (200 mg/kg/day). These signs were considered of no toxicological relevance.
Found dead and/or missing pups were observed both in control and treated groups, with similar incidence.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences were noted in thyroid weight between control and treated pups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Autolysed abdominal and/or thoracic organs were generally observed in pups found dead at birth and/or in those which died during the lactation period.
No significant signs were recorded in pups sacrificed on Days 4 or 14 post partum. Tip of tail missing was observed in two pups (one of Group 2 and one of Group 3), in the pup of Group 2 also swollen left hindlimb was noted. In addition, in all pups of dam no. X0690043 (Group 3), hairloss on the head was recorded.
No nipples were observed in male pups on Day 14 post partum.

Details on results (F1)

LITTER DATA AT BIRTH, ON DAYS 1 AND 4 POST PARTUM (BEFORE CULLING) AND ON DAY 13 POST PARTUM (AFTER CULLING) AND SEX RATIO OF PUPS
Litter data of treated females recorded at birth, on Days 4 and 13 post partum, were comparable to the control group.
Sex ratios at birth, on Days 4 and 13 post partum, did not show differences between groups, when calculated as the percentage of males.

THYROID HORMONES IN PUPS - DAY 14 POST PARTUM
Thyroid stimulating hormone (TSH) was increased in some female pups of the high dose group. Mean group data were 54 % above controls. Due to the absence of other related changes, this finding was considered of no toxicological relevance.

ANOGENITAL DISTANCE
No relevant differences were seen between the control and treated pups in anogenital distance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation and gave birth on Day 22 post coitum (mean value). Litter data and sex ratios were unaffected by treatment. Similar clinical signs were recorded in control and treated pups during the lactation period. No relevant differences were seen between the control and treated pups in anogenital distance. Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect, as well as in thyroid weight of pups sacrificed on Day 14 post partum.

Hormone analysis did not reveal any alterations in parental males. Thyroid stimulating hormone was increased in a number of female pups at dose level of 800 mg/kg/day. Due to the absence of other related changes, this finding was considered of no toxicological relevance.

Terminal body weight and organ weights did not show relevant differences between control and treated groups.

No treatment-related changes were noted at macroscopic and microscopic observations, as well as on the evaluation of the seminiferous tubules respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

Table 1: Pregnancy status.

Group:	1	2	3	4
non-pregnant females	0	2	1	0
unilateral total resorption	0	0	0	1
no. of females at term with live pups	10	8	9	9

Table 2: Fate of females - group incidence.

Group:	1	2	3	4
Initial group size	10	10	10	10
Not pregnant	0	2	1	0
Unilateral total resorption	0	0	0	1
With live foetuses at gestation on day 20	10	8	9	9

Table 3: Clinical signs of males - group incidence.

Group	1		2		3		4
No. animals	10		10		10		10
	no. animals	no. days	no. animals	no. days	no. animals	no. days	no. animals	no. days
No significant signs	10	29.0	10	29.0	10	29.0	10	14.1
Appearance
Staining	0	0.0	0	0.0	0	0.0	9	14.1

Table 4: Clinical signs of females before pairing - group incidence.

Group	1		2		3		4
No. animals	10		10		10		10
	no. animals	no. days	no. animals	no. days	no. animals	no. days	no. animals	no. days
No significant signs	10	15.0	10	15.0	10	15.0	10	10.2
Appearance
Staining	0	0.0	0	0.0	0	0.0	5	9.6

Table 5: Clinical signs of femalespost coitumandpost partumperiods - group incidence.

Group	1		2		3		4
No. animals	10		10		10		10
	no. animals	no. days	no. animals	no. days	no. animals	no. days	no. animals	no. days
Appearance
Staining	0	0.0	0	0.0	7	70.0	10	100.0
Hair loss	0	0.0	0	0.0	0	0.0	1	10.0

Table 6: Mean body weight (g) of males before pairing and from pairing period to sacrifice.

Group		Day (before pairing)				Day (from pairing period to sacrifice)
		1 (pretest phase)	1 (premating phase)	8	15 (start of mating phase)	8	15
1	no. animals	10	10	10	10	10	10
	mean	328.36	344.85	353.94	371.03	375.99	395.51
	s.d.	10.46	13.45	13.72	16.66	16.37	18.18
2	no. animals	10	10	10	10	10	10
	mean	327.50	344.45	354.14	368.62	375.76	393.41
	s.d.	12.06	14.20	11.18	14.43	14.69	17.45
3	no. animals	10	10	10	10	10	10
	mean	327.96	346.29	354.05	366.74	371.72	392.05
	s.d.	11.34	16.66	20.16	20.59	20.03	29.24
4	no. animals	10	10	9	9	9	9
	mean	328.15	339.95	354.70	371.29	376.70	393.79
	s.d.	11.34	15.28	15.78	16.92	16.86	15.45

Table 7: Mean body weight (g) of females before pairing and during post coitum and post partum periods.

Group		Day (before pairing)				Day (post coitumandpost partumperiods)
		1 (pretest phase)	1 (premating phase)	8	15 (start of mating phase)	0 (gestation phase)	7	14	20	1 (post partumphase)	4	7	13
1	no. animals	10	10	10	10	10	10	10	10	10	10	10	10
	mean	232.22	239.17	238.11	246.46	249.04	276.05	306.59	392.17	295.10	304.04	314.04	325.33
	s.d.	6.62	8.13	9.83	10.78	12.99	11.71	10.41	16.03	11.36	8.43	11.00	12.11
2	no. animals	10	10	10	10	8	8	8	8	8	8	8	8
	mean	232.25	240.26	241.51	252.40	250.28	272.60	301.68	377.40	292.79	302.74	304.40	323.32
	s.d.	7.25	9.42	10.17	8.87	8.57	9.75	8.40	27.20	9.78	10.35	5.39	8.30
3	no. animals	10	10	10	10	9	9	9	9	9	9	9	9
	mean	231.63	237.97	234.09	242.81	244.34	271.11	301.36	384.34	294.81	302.35	308.55	323.51
	s.d.	6.97	8.09	8.99	10.06	8.80	10.50	9.49	14.26	11.28	10.40	13.71	10.89
4	no. animals	10	10	10	9*	10	10	10	10	9	9	9	9
	mean	232.39	247.37	245.74	247.94	249.33	277.19	306.30	367.66	297.25	310.80	313.79	327.32
	s.d.	7.24	9.66	11.59	11.98	10.60	13.88	13.17	44.66	13.49	14.44	13.55	11.04

* one female excluded from group mean data as the pairing phase started on day 16

Table 8: Mean body weight (g) gain of males before pairing and from pairing to sacrifice.

Group		Day (before pairing)			Day (from pairing period to sacrifice)
		1 (premating phase)	8	15 (start of mating phase)	8	15
1	no. animals	10	10	10	10	10
	mean	2.355	1.299	2.442	0.708	2.788
	s.d.	0.759	0.792	0.779	0.533	0.795
2	no. animals	10	10	10	10	10
	mean	2.422	1.384	2.070	1.020	2.521
	s.d.	0.795	0.933	0.739	0.279	1.597
3	no. animals	10	10	10	10	10
	mean	2.620	1.108	1.812	0.712	2.904
	s.d.	0.856	0.766	0.534	0.355	1.777
4	no. animals	10	9	9	9	9
	mean	1.686	1.837	2.370	0.773	2.441
	s.d.	0.964	0.432	0.647	0.153	0.574

Table 9: Mean body weight (g) gain of females before pairing and during post coitum and post partum periods.

Group		Day (before pairing)			Day (from pairing period to sacrifice)
		1 (premating phase)	8	15 (start of premating phase)	7 (gestation phase)	14	20	4 (post partumphase)	7	13
1	no. animals	10	10	10	10	10	10	10	10	10
	mean	0.993	-0.151	1.193	3.859	4.363	14.262	3.142	3.171	1.883
	s.d.	0.921	0.741	0.646	0.918	0.710	1.396	2.245	2.088	1.358
2	no. animals	10	10	10	8	8	8	8	8	8
	mean	1.145	0.178	1.556	3.189	4.155	12.619	3.319	0.552	3.154
	s.d.	0.874	1.120	0.617	1.225	0.788	3.716	1.899	2.516	1.511
3	no. animals	10	10	10	9	9	9	9	9	9
	mean	0.906	-0.554	1.246	3.824	4.322	13.829	2.512	2.066	2.494
	s.d.	0.752	0.756	0.846	1.325	0.674	1.752	1.308	2.622	0.991
4	no. animals	10	10	9*	10	10	10	9	9	9
	mean	2.141**	-0.233	0.394	3.980	4.158	10.226	4.517	0.996	2.255
	s.d.	1.236	0.744	1.077	1.452	1.302	5.815	1.185	2.336	1.361

* one female excluded from group mean data as the pairing phase started on day 16

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

Table 10: Mean food consumption of males before pairing (g/animal/day).

Group		Day (before pairing)			Day (post coitumandpost partumperiods)
		8 (pretest phase)	8 (premating phase)	15	7 (gestation phase)	14	20	7 (post partumphase)	13
1 (male)	no. animals	2	2	2
	mean	22.27	22.23	22.58
2 (male)	no. animals	2	2	2
	mean	22.17	23	20.34
3 (male)	no. animals	2	2	2
	mean	21.88	22.5	21.77
4 (male)	no. animals	2	2	2
	mean	21.84	22.03	23.04
1 (female)	no. animals	2	2	2	10	10	10	10	10
	mean	16.69	16.08	16.73	21.66	21.84	26.48	41.02	58.37
	s.d.				1.83	1.44	4.85	3.70	3.73
2 (female)	no. animals	2	2	2	8	8	8	8	8
	mean	16.05	16.83	16.86	20.02	23.63	25.03	37.55	53.13
	s.d.				4.01	6.22	2.15	3.85	8.07
3 (female)	no. animals	2	2	2	9	9	9	9	9
	mean	15.87	15.62	16.60	21.27	22.26	26.21	41.17	55.17
	s.d.				1.71	1.16	1.94	6.51	7.55
4 (female)	no. animals	2	2	2	10	10	10	9	9
	mean	17.02	17.36	17.03	21.15	23.24*	26.63	41.16	58.01
	s.d.				3.23	1.00	2.74	3.85	3.98

* mean value is significantly different from control at p < 0.05; modified t test if group variances are inhomogeneous

Table 11: Mean thyroid hormone determination in parental males.

Group:		1	2	3	4
Triiodothyronine (ng/ml)	mean	9.845	9.011	9.15	9.396
	s.d.	1.024	1.092	1.322	1.472
	no.	10	10	10	9
Thyroxine (ng/ml)	mean	321	352.5	329.5	316.4
	s.d.	29.1	75.4	31.1	26.3
	no.	10	10	10	9
Thyroid stimulating hormone (ng/ml)	mean	2.428	2.331	1.986	2.247
	s.d.	1.59	1.081	1.239	0.894
	no.	10	10	10	9

Table 12: Mean thyroid hormone determination in male and female pups on day 14post partum.

Group:		Males				Females
		1	2	3	4	1	2	3	4
Triiodothyronine (ng/ml)	mean	6.638	6.086	5.794	6.139	6.364	5.830	6.860	6.632
	s.d.	1.008	1.414	0.748	1.309	1.654	0.686	2.165	2.078
	no.	9	8	9	9	10	8	9	9
Thyroxine (ng/ml)	mean	184.5	187.6	194.3	199.4	211.9	205.2	229.6	234.3
	s.d.	13.5	16.4	20.8	20.8	17.5	41.1	27.4	24.1
	no.	10	8	9	9	10	8	9	9
Thyroid stimulating hormone (ng/ml)	mean	1.384	1.706	1.511	1.550	1.174	1.578	1.476	1.810*
	s.d.	0.610	1.001	0.521	0.437	0.320	0.396	0.664	0.738
	no.	10	8	9	9	10	8	9	9

Table 13: Oestrus cycle before pairing - group summary data.

Animal no.	Group 1 oestrus cycles	Animal no.	Group 2 oestrus cycles	Animal no.	Group 3 oestrus cycles	Animal no.	Group 4 oestrus cycles
1	3	21	3	41	3	61	1
3	2	23	3	43	3	63	3
5	2	25	3	45	3	65	2
7	1	27	1	47	2	67	1
9	3	29	1	49	2	69	1
11	3	31	2	51	2	71	2
13	2	33	0	53	4	73	3
15	2	35	3	55	2	75	3
17	2	37	1	57	4	77	2
19	3	39	3	59	3	79	3

Table 14: Summary of reproductive parameters of animals.

Group:	Male				Female
	1	2	3	4	1	2	3	4
Copulatory index (%)*	100.0	100.0	100.0	100.0	100.0	100.0	100.0	100.0
Fertility index (%)**	100.0	80.0	90.0	100.0	100.0	80.0	90.0	100.0

* copulatory index (%) = no. animals mated / no. animals paired × 100

** fertility index (%) = no. pregnant females / no. males paired × 100

Table 15: Mean implantation, pre-implantation loss, pre-birth loss and gestation length data.

Group		Corpora lutea	Implantations	Pre-implantation loss (%)	Total litter size at birth	Pre-natal loss (%)	Gestation length (days)
1	mean	15.00	15.00	0.00	14.40	3.73	22.40
	s.d.	2.00	2.00	0.00	1.78	5.18	0.52
	no. animals	10	10	10	10	10	10
2	mean	13.75	13.75	0.00	13.00	6.49	22.38
	s.d.	4.71	4.71	0.00	4.69	6.19	0.52
	no. animals	8	8	8	8	8	8
3	mean	14.44	14.44	0.00	13.56	7.18	22.56
	s.d.	2.74	2.74	0.00	3.28	11.36	0.53
	no. animals	9	9	9	9	9	9
4	mean	14.22	14.22	0.00	13.11	7.60	22.44
	s.d.	2.73	2.73	0.00	2.98	11.90	0.73
	no. animals	9	9	9	9	9	9

Table 16: Mean litter data at birth on days 1 and 4 post partum before culling.

Group		At birth			Day 1post partum			Day 4post partum				Day 13post partum
		total litter size	live litter size	pup loss (%)	total litter size	litter weight (g)	mean pup weight (g)	live litter size	cumulative loss (%)	litter weight (g)	mean pup weight (g)	live litter size	post natal loss (%)	litter weight (g)	mean pup weight (g)
1	mean	14.40	13.60	5.67	13.00	88.51	6.80	12.90	10.57	126.10	9.89	7.80	0.00	236.50	30.27
	s.d.	1.78	2.12	7.76	3.20	21.34	0.66	3.14	18.01	26.69	1.06	0.63	0.00	26.52	1.59
	no. animals	10	10	10	10	10	10	10	10	10	10	10	10	10	10
2	mean	13.00	11.88	7.93	11.75	76.94	6.73	11.63	11.84	110.90	10.04	6.88	1.56	204.40	30.39
	s.d.	4.69	4.19	5.64	4.46	26.90	0.74	4.41	7.80	34.27	1.62	1.89	4.42	26.52	1.59
	no. animals	8	8	8	8	8	8	8	8	8	8	8	8	8	8
3	mean	13.56	13.00	4.08	12.67	88.16	7.01	12.56	7.58	122.80	10.07	7.22	1.39	221.20	30.80
	s.d.	3.28	3.28	6.76	3.57	25.22	0.76	3.54	12.09	31.30	1.61	1.30	4.17	42.03	3.29
	no. animals	9	9	9	9	9	9	9	9	9	9	9	9	9	9
4	mean	13.11	12.78	3.17	12.44	84.29	6.86	12.33	6.49	119.60	9.92	7.56	0.00	231.80	30.91
	s.d.	2.98	3.27	7.44	3.36	21.95	0.86	3.28	8.49	25.98	1.33	0.88	0.00	19.91	2.85
	no. animals	9	9	9	9	9	9	9	9	9	9	9	9	9	9

Table 17: Mean sex ratios of pups.

Group		At birth				Day 4post partum				Day 13post partum
		M	F	Total	% males	M	F	Total	% males	M	F	Total	% males
1	mean	7.00	7.40	14.40	48.61	6.10	6.80	12.90	48.69	4.00	3.80	7.80	51.67
	s.d.	1.70	1.71	1.78	9.41	1.52	2.49	3.14	10.28	0.00	0.63	0.63	5.28
	no. animals	10	10	10	10	10	10	10	10	10	10	10	10
2	mean	6.38	6.63	13.00	48.21	5.75	5.88	11.63	50.99	3.50	3.38	6.88	52.66
	s.d.	3.34	2.88	4.69	13.43	2.82	3.14	4.41	15.18	0.76	1.30	1.89	8.69
	no. animals	8	8	8	8	8	8	8	8	8	8	8	8
3	mean	7.67	5.89	13.56	56.78	6.89	5.67	12.56	53.86	3.67	3.56	7.22	51.43
	s.d.	2.24	2.42	3.28	11.48	2.76	2.06	3.54	12.27	0.50	0.88	1.30	5.38
	no. animals	9	9	9	9	9	9	9	9	9	9	9	9
4	mean	5.78	7.33	13.11	43.59	5.33	7.00	12.33	42.37	3.67	3.89	7.56	48.14
	s.d.	1.79	1.80	2.98	8.27	1.94	1.80	3.28	8.50	0.71	0.33	0.88	5.57
	no. animals	9	9	9	9	9	9	9	9	9	9	9	9

Table 18: Mean angiogenital distance of males and females.

Group		Male				Female
		1	2	3	4	1	2	3	4
Angiogenital distance (normalised) (mm/g/3)	no. animals	61	47	64	48	69	47	50	64
	mean	2.16	2.21	2.21	2.22	1.26	1.33	1.24	1.26
	s.d.	0.31	0.25	0.22	0.18	0.26	0.19	0.19	0.15

Table 19: Mean absolute body weights of males and females.

Group		Male				Female
		1	2	3	4	1	2	3	4
Terminal body weight (g)	no. animals	10	10	10	9	10	10	10	10
	mean	396.97	393.58	391.22	393.01	324.74	316.45	313.07	322.66
	s.d.	17.53	17.70	27.60	16.05	10.58	17.39	19.42	20.87
	p < 0.05		22.35	22.35	22.96		19.21	19.21	19.21
	p < 0.01		28.36	28.36	29.13		24.36	24.36	24.36

Table 20: Mean absolute organ weights of males and females (data homogeneous by Bartlett's test (Dunnet's test).

Group		Male				Female
		1	2	3	4	1	2	3	4
Epididymes	no. animals	10	10	10	9
	mean	1.3434	1.3611	1.2951	1.2324
	s.d.	0.1330	0.1997	0.1315	0.0832
	p < 0.05		0.1585	0.1585	0.1628
	p < 0.01		0.2011	0.2011	0.2066
Prostate	no. animals	10	10	10	9
	mean	1.2217	1.1403	1.0632	1.0591
	s.d.	0.2684	0.1460	0.1708	0.2453
	p < 0.05		0.2336	0.2336	0.2400
	p < 0.01		0.2964	0.2964	0.3045
Seminal vesciles	no. animals	10	10	10	9
	mean	1.7436	1.4737	1.6823	1.5083
	s.d.	0.4468	0.3391	0.2538	0.3760
	p < 0.05		0.3955	0.3955	0.4063
	p < 0.01		0.5019	0.5019	0.5157
Spleen	no. animals	10	10	10	9	10	10	10	10
	mean	0.8003	0.8282	0.8080	0.7658	0.7075	0.6760	0.6901	0.6796
	s.d.	0.0636	0.0695	0.1009	0.0632	0.0781	0.0615	0.0523	0.0710
	p < 0.05		0.0837	0.0837	0.0860		0.0728	0.0728	0.0728
	p < 0.01		0.1062	0.1062	1091.0000		0.0924	0.0924	0.0924
Testes	no. animals	10	10	10	9
	mean	3.7640	3.7207	3.7271	3.6967
	s.d.	0.2679	0.3178	0.2370	0.2049
	p < 0.05		0.2873	0.2873	0.2951
	p < 0.01		0.3645	0.3645	0.3745
Thymus	no. animals	10	10	10	9	10	10	10	10
	mean	0.3739	0.5544	0.3826	0.3793	0.2135	0.2499	0.2667	0.2147
	s.d.	0.0689	0.6125	0.0635	0.0712	0.0461	0.0988	0.1020	0.0585
	p < 0.05		0.4409	0.0670	0.0736		0.0879	0.0801	0.0533
	p < 0.01		0.6329	0.0962	0.1064		0.1115	0.1149	0.0765
Thyroid	no. animals	10	10	10	9	10	10	10	10
	mean	0.0273	0.0266	0.0269	0.0247	0.0259	0.0221	0.0235	0.0266
	s.d.	0.0029	0.0052	0.0034	0.0247	0.0067	0.0034	0.0026	0.0034
	p < 0.05		0.0040	0.0040	0.0041		0.0055	0.0051	0.0054
	p < 0.01		0.0051	0.0051	0.0052		0.0079	0.0074	0.0077
Ovaries	no. animals					10	10	10	10
	mean					0.1257	0.1333	0.1315	0.1208
	s.d.					0.0175	0.0316	0.0157	0.0214
	p < 0.05						0.0246	0.0246	0.0246
	p < 0.01						0.0312	0.0312	0.0312
Uterus	no. animals					10	10	10	10
	mean					0.3597	0.4635	0.4041	0.4291
	s.d.					0.0483	0.3139	0.0990	0.2026
	p < 0.05						0.2272	0.0788	0.1490
	p < 0.01						0.3260	0.1131	0.2137

Table 21: Mean organ weight to terminal body weight ratios (%) of males and females (data homogeneous by Bartlett's test (Dunnet's test).

Group		Male				Female
		1	2	3	4	1	2	3	4
Epididymes	no. animals	10	10	10	9
	mean	0.3382	0.3462	0.3329	0.3138
	s.d.	0.0279	0.0511	0.0445	0.0203
	p < 0.05		0.0422	0.0422	0.0433
	p < 0.01		0.0535	0.0535	0.0550
Prostate	no. animals	10	10	10	9
	mean	0.3078	0.2909	0.2722	0.2707
	s.d.	0.0664	0.0443	0.0415	0.0663
	p < 0.05		0.0610	0.0610	0.0626
	p < 0.01		0.0774	0.0774	795.0000
Seminal vesciles	no. animals	10	10	10	9
	mean	0.4399	0.3757	0.4325	0.3844
	s.d.	0.1125	0.0917	0.0766	0.0982
	p < 0.05		0.1049	0.1049	0.1078
	p < 0.01		0.1331	0.1331	0.1368
Spleen	no. animals	10	10	10	9	10	10	10	10
	mean	0.2020	0.2105	0.2072	0.1951	0.2180	0.2143	0.2212	0.2108
	s.d.	0.0186	0.0168	0.0284	0.0178	0.0246	0.0246	0.0215	0.0179
	p < 0.05		0.0231	0.0231	0.0237		0.0241	0.0241	0.0241
	p < 0.01		0.0293	0.0293	0.0301		0.0306	0.0306	0.0306
Testes	no. animals	10	10	10	9
	mean	0.9489	0.9460	0.9594	0.9414
	s.d.	0.0642	0.0769	0.1179	0.0549
	p < 0.05		0.0908	0.0908	0.0933
	p < 0.01		0.1152	0.1152	0.1184
Thymus	no. animals	10	10	10	9	10	10	10	10
	mean	0.0943	0.1411	0.0978	0.0966	0.0656	0.0796	0.0851	0.0665
	s.d.	0.0169	0.1550	0.0146	0.0183	0.0130	0.0326	0.0313	0.0173
	p < 0.05		0.1115	0.0160	0.0186		0.0251	0.0243	0.0155
	p < 0.01		0.1600	0.0230	0.0268		0.0360	0.0348	0.0222
Thyroid	no. animals	10	10	10	9	10	10	10	10
	mean	0.0069	0.0067	0.0069	0.0063	0.0080	0.0071	0.0075	0.0083
	s.d.	0.0007	0.0012	0.0008	0.0005	0.0022	0.0013	0.0009	0.0012
	p < 0.05		0.0010	0.0010	0.0010		0.0018	0.0017	0.0018
	p < 0.01		0.0012	0.0012	0.0012		0.0027	0.0024	0.0026
Ovaries	no. animals					10	10	10	10
	mean					0.0387	0.0426	0.0422	0.0377
	s.d.					0.0054	0.0126	0.0062	0.0074
	p < 0.05						0.0098	0.0059	0.0065
	p < 0.01						0.0141	0.0084	0.0094
Uterus	no. animals					10	10	10	10
	mean					0.1108	0.1500	0.1298	0.1366
	s.d.					0.0149	0.1116	0.0344	0.0789
	p < 0.05						0.0805	0.0344	0.0789
	p < 0.01						0.1156	0.0385	0.0824

Table 22: Macroscopic observations - group incidence.

Died by unscheduled death
Group:		4 (male)
No. animals in group:		1
Liver	abnormal shape	1
Thymus	abnormal colour	1
Aorta	abnormal colour	1
Diaphragm	abnormal colour	1
Heart	abnormal colour	1
Lungs	abnormal colour	1
Sternum	abnormal colour	1
Thoracic cavity	abnormal contents	1
Died at final sacrifice
Group:		Males				Females
		1	2	3	4	1	2	3	4
No. animals in group:		10	10	10	9	10	10	10	10
Ovaries	abnormal size	-	-	-	-	0	1	0	0
Thymus	abnormal colour	0	0	0	0	0	1	0	1
	abnormal size	0	0	0	0	3	4	4	4
Uterus	abnormal size	-	-	-	-	0	1	0	0
	abnormal contents	-	-	-	-	0	1	0	0
	not pregnant	-	-	-	-	0	2	1	0
	unilateral total resorption	-	-	-	-	0	0	0	1
Forelimbs	hair loss	0	0	0	0	0	0	0	1
Tail	staining	0	0	0	2	0	0	0	4
Whole animal	no abnormalities	10	10	10	7	7	4	5	3

Table 23: Microscopic observations.

Died by unscheduled death
Group:		4 (male)
No. animals in group:		1
Kidneys	nephropathy	0
	mineralisation	0
	congestion/haemorrhage	1
Liver	inflammatory cell foci	0
	clear cell change	0
	congestion	1
Testes	germ cell degeneration	1
Thymus	atrophy	0
	congestion	0
	haemorrhage	1
Lungs	chronic inflammation	1
	foreign material	1
	necrosis	1
Heart	inflammatory cell foci	1
	congestion/haemorrhage	1
	myocardial necrosis	1
Died at final sacrifice
Group:		Males				Females
		1	2	3	4	1	2	3	4
No. animals in group:		10	10	10	9	10	10	10	10
Adrenals	cortical cell vacuolisation	1	-	-	1	0	-	-	0
	accessory nodule	0	-	-	0	2	-	-	1
	cortical hypertrophy	0	-	-	0	1	-	-	1
Kidneys	nephropathy	8	-	-	8	3	-	-	4
	mineralisation	0	-	-	0	0	-	-	1
Liver	inflammatory cell foci	3	-	-	2	0	-	-	2
	clear cell change	2	-	-	3	6	-	-	2
	congestion	0	-	-	0	0	-	-	0
Testes	germ cell degeneration	1	-	-	0	-	-	-	-
Thyroid	thyro-glossal duct remnant	0	-	-	1	0	-	-	0
Uterus	luminal dilatation	-	-	-	-	0	1*	0	0
Thymus**	atrophy	-	-	-	-	2	4	4	4
	congestion	-	-	-	-	0	0	0	1

* out of 1 animal tested

** no males tested; 3, 4, 4 and 5 females tested from groups 1, 2, 3 and 4

Applicant's summary and conclusion

Conclusions:

The NOAEL for general toxicity and for fertility and reproduction parameters was considered to be 800 mg/kg bw/day for males and females.

Executive summary:

In order to provide information on toxicity as well as any possible effects of the test item on male and female reproductive performance, a study was performed according to the OECD Guideline 421 (2016). The effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring were investigated.

All doses (0, 50, 200 and 800 mg/kg/day) were administered orally, by gavage to 10 males and 10 females per dose group. The control group received softened water (by reverse osmosis). The dose volume was 10 mL/kg bodyweight. Males were treated for 2 weeks prior to pairing and during pairing with females until theday before necropsy, for a total of 28 or 29 days. Females were treated for 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). Four not pregnant females were dosed up to the day before necropsy.

The following investigations were performed: mortality check, clinical signs, body weight, food consumption, oestrous cycle, mating performance, litter data, sex ratios, macroscopic observations and organ weights.

Histopathological examination was performed on control and high dose groups, on all abnormalities detected during post mortem observation from all groups and on one found dead male.

The identification of the stages of the spermatogenic cycle was also performed in all males of the control and high dose groups. Thyroid hormone determination was performed in all parental males. Clinical signs were recorded in all pups. Thyroid weight and thyroid hormone determination of 1 pup/sex/litter (where possible) at Day 14 post partum were determined. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was confirmed by internal gonads inspection. All live pups sacrifice on Day 14 post partum were examined for extenal abnormalities and sex confirmation by gonadal inspection. The presence of nipples/areolae was verified in male pups.

Mortality and fate of females

One male from the high dose group was found dead on Day 7 of the pre-mating phase of the study. No clinical signs were observed prior to death. On the basis of the macroscopic (dark fluid contents in the thoracic cavity and dark colour of lungs) and microscopic observation (essentially chronic inflammation and necrosis in the lungs with brown foreign material, test item like, and brown pigment-laden macrophages in the alveoli of the lungs), the cause of death was attributed to a misdosing.

Two females receiving 50 mg/kg/day and one female receiving 200 mg/kg/day were proved not pregnant at necropsy. In addition, one female receiving 800 mg/kg/day had unilateral total resorption in the left horn and was not pregnant in the right one. The number of females with live pups on Day 14 post partum was: 10 in the control, 8 in the low dose (50mg/kg/day), 9 in the mid-dose (200mg/kg/day) and 9 in the high dose group (800mg/kg/day).

Clinical signs

No relevant clinical signs were observed during the study. Staining observed in high dose group of both sexes and in females of mid-dose group was considered related to the colour of the test item.

Observations of the cage tray

Staining in the cage tray and red faeces generally observed in mid- and/or high dose groups were considered related to the colour of the test item which was eliminated by the animals.

Body weight and body weight gain

No relevant differences in body weight and body weight gain were noted between control and treated males and females throughout the study.

Food consumption

No effects on food consumption were observed in both sexes.

Thyroid hormone determination

Parental males

No changes in thyroid hormones were recorded in parental male animals of all treated groups.

Pups – Day 14 post partum

Changes observed in thyroid stimulating hormone of some female pups of the high dose group were considered of no toxicological relevance due to the absence of other related changes.

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females

No significant differences were observed for these parameters between treated and control groups. All pregnant dams gave birth on Day 22 post coitum (mean value).

Litter data at birth, on Days 1 and 4 post partum (before culling) and on Day 13 post partum (after culling) and sex ratio of pups

Litter data at birth, on Days 4 and 13 post partum recorded on treated groups were comparable to those of the control group. Sex ratios at birth, on Days 4 and 14 post partum did not show differences between groups, when calculated as the percentage of males.

Clinical signs of pups

Pre-weaning clinical signs were comparable between treated and control groups.

Anogenital distance

No relevant differences were seen between the control and treated pups in anogenital distance.

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 14 post partum and nipple count

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups on Day 14 post partum.

Pups thyroid weight on Day 14 post partum

No differences were noted in thyroid weight between control and treated pups.

Terminal body weight and organ weights

Terminal body weight and organ weights did not show relevant differences between control and treated groups.

Macroscopic observations

No remarkable differences were noted at post mortem examination in treated animals sacrificed at the end of the study, when compared with controls.

Microscopic observations

No treatment-related changes were observed in treated animals sacrificed at the end of the study, when compared to the controls.

Conclusion

On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and for fertility and reproduction parameters was considered to be 800 mg/kg/day for males and females.