3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25. Nov 2021 - 31. Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25. Nov 2021 - 31. Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch no.: 0004460012
Purity: 99.3 wt%
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: Room temperature

The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to a quality system.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hannover

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany, D-97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8-10 weeks
- Weight at study initiation: (P) Males: 208-227 g; Females: 198 -217 g
- Housing: 5 of one sex to a cage until pairing; 1:1 (m/f) during pairing; after pairing: females individually, males as before mating
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum
- Acclimation period: 2 weeks

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

IN-LIFE DATES: From: 25 Nov 2021 To: Feb 2022 (females), Jan 2022 (males)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The preparations were made weekly and every three days at concentration of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
A 28 hour stability at room temperature and an 8 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in internal SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the formulations prepared on the first and the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in internal SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC.

Duration of treatment / exposure:

Males: 34/35 d
Females: 66 d

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Medium dose group

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), at least once daily (clinical signs)
- Cage side observations: mortality, clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, additionally females on days 0, 7, 14 and 20 p.c.; Dams on days 1, 4, 7 and 13 p.p. and prior necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food consumed by each cage of males and females was recordedweekly during the pre-mating period starting from the beginning of treatment up to mating. Individual food consumption for mated females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

PARTUITION CHECK:
- three times a day during the working day and twice daily during the weekends and Public Holidays
- from Day 20 to Day 25 post coitum

BLOOD COLLECTION:
- Males: Blood samples (approximately 0.8 mL) were collected on the day of sacrifice under isoflurane anaesthesia from the sublingual vein.
- Females: As part of the necropsy procedure, blood samples (approximately 0.8 mL) were withdrawn under isoflurane anaesthesia from the abdominal vena cava.

IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH)

Oestrous cyclicity (parental animals):

The assessment of oestrous cycles, by vaginal smears, was performed once daily in the morning in stock females and in allocated females for 2 weeks before the start of dosing.
From Day 1 of treatment, vaginal smears were taken in the morning from all females throughout the mating period until a positive identification of copulation was detected.

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 and the oestrous cycle phase recorded.

Litter observations:

STANDARTISATION OF LITTER:
- on Day 4 post partum,
- litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter
- Partial adjustment (for example, 5 males and 3 females) was acceptable
- no pups were eliminated when litter size dropped below the culling target (8 pups/litter).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths, live births
- weight gain (weighing on day 1, 4, 7, 13)
- postnatal mortality
- presence of gross anomalies
- anogenital distance (AGD)
- pup weight on the day of AGD
- presence of nipples/areolae in male pups (Day 13/14)

GROSS EXAMINATION OF DEAD PUPS

BLOOD COLLECTION:
- On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 mL (per sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).
In order to obtain the required volume, blood samples were pooled from two or three pups on Day 4 post partum.

IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, on Day 21/22 of the mating phase, after 34/35 days of treatment
- Maternal animals: The dams with live pups were killed on Day 14 post partum. One female showing no evidence of copulation was sacrificed 28 days after the last day of the mating session (Day 43 of the mating phase). The five females which did not give birth 25 days after positive identification of mating were killed shortly after. Two control females dnd one mid-dose female were sacrificed on Day 28 of the gestation phase. One mid-dose female and one high dose female were sacrificed on Day 27 post coitum.

NECROPSY
- The clinical history of the males and females of the parental generation 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 and the required tissue samples preserved in fixative and processed for histopathological examination.
- All females were also examined for the number of visible implantation sites (pregnant animals). Uteri from females with no visible implantations, and uterus from one female with implantations only on the right horn, were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
Pups that had completed the scheduled test period (Day 4 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

GROSS NECROPSY
- The pup from one dam (Group 3) found dead in the cage was examined for external and internal abnormalities.
- All culled pups sacrificed at 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 examined for external abnormalities and sex confirmation by gonadal inspection.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Thyroid

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:

Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day when the parturition is defined complete (Day 0 post partum).

Males:

Copulatory Index (%) = no. of males with confirmed mating/no. of males cohabitated ×100

Fertility Index (%) = no. of males which induced pregnancy/no. of males cohabitated ×100

Females:

Copulatory Index (%) = no. of females with confirmed mating/no. of females cohabitated ×100

Fertility Index (%) = no. of pregnant females/no. of females cohabitated ×100

Pup loss at Day 0 post partum was calculated as a percentage from the formula:
(Total litter size−Live litter size)/Total litter size ×100

Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations−Live litter size at birth)/no. of visible implantations ×100

Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from
the formula:
(Live litter size at birth−live litter size at Day 4 (before culling))/Live litter size at birth ×100

Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from
the formula:
(Live litter size onDay 4 (after culling)−Live litter size onDay 13)/Live litter size onDay 4 (after culling) ×100

Males and females:

Pre coital Interval = The number of nights paired prior to the detection of mating

Offspring viability indices:

Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and was presented as the percentage of males per litter.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences in body weight were recorded between treated and control groups throughout the entire study.
A degree of variability in body weight gain was observed in both males and females over the course of the study and among all groups, but no relationship with the test item treatment was observed. Notably, there was a -58% decrease in body weight gain in high dose females at Day 7 post partum, compared to the control, but without statistical significance.
Being isolated event and without a dose-related trend, this case was not considered to be treatment related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

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:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The oestrous cyclicity and the number of copulatory plug (mean value) of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment.
The mean number of oestrous cycles observed in treated animals and controls was comparable and was of 3/4 cycles. The mean number of copulatory plugs found in the cage was 3 in control, mid and high dose groups, and 4 in low dose group.
Pre-coital interval, copulatory and fertility indices were considered to be unaffected by administration of the test item. The copulatory indices were 90% for controls and 100% in all treated groups of both sexes. The fertility indices in males and females were 70% for controls, 100% in low dose animals and 90% in mid- and high dose animals.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The number of implantations, live litter size and the percentage of prenatal loss did not
show treatment-related differences. One female in the mid-dose group had total
resorptions.
Mean gestation period was comparable between control and treated animals.
All dams give birth between Days 22 and 23 post coitum.
The slight differences noted in mean values of total litter size, live litter size at birth and on Days 1, 4 and 13 post partum and litter weight were without a dose-related trend nor statistical significance, therefore considered not related to treatment. Moreover, these data remained almost unchanged within each group throughout the post partum phase.
Post natal loss was observed in low and mid- dose groups: one low dose female and two mid-dose females which lost their pups (missing or found dead) respectively on Day 7 and on Day 4 post partum. On statistical analysis, the mean results of the low and mid-dose groups were found to be significant on these days, but being single cases and in the absence of a dose-related trend, they were not considered to be caused by test item treatment.
The mean pup weight at all stages was always comparable between groups or even higher for pups from treated females respect to the controls.
Sex ratios at birth, on Days 4 and 14, expressed as percentage of males, did not show significant differences between groups.

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic, reproductive and developental toxicity

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

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were some missing pups from low (one pup) and mid-dose group (three pups), and one pup found dead from one mid-dose female.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

one thin pup from one control dam seen only at Day 13 post partum.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

No nipples in males were noted.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significance decrease in mean thyroid weight was found in male pups of mid-dose group respect to than from control dams. This decrease (-21%) was slight and not dose related, therefore considered incidental. No other statistically significant changes were noted.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 2: Reproduction data

Groups	1	2	3	4
Paired No.	10	10	10	10
Positive identification of mating No.	9	10	10	10
Not pregnant	3	0	1	1
Conceiving number	7	10	9	9
Total resorptions	0	0	1	0
Total litter loss	0	0	0	0
Positive identification of mating 1 - 5 days	9	9	8	7
Positive identification of mating 6 - 14 days	0	1	2	3
No. of females with live pups on Day 14 post partum	7	10	8	9

 

Table 3: Reproduction data and litter data - group means

Summary of Reproduction Data
	Dosage levels mg/kg/day
Observations	0	100	300	1000
No. Dams Inseminated	9	10	10	10
Oestrus cycle (mean number)	4	3	4	4
No. Dams Pregnant	7	10	9	9
Percent Dams Pregnant	70.0	100.0	90.0	90.0
Copulatory interval (1-5 days)	9	9	8	7
Copulatory interval (6-14 days)	0	1	2	3
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	1	0
No. of Litters	7	10	8	9
Total No. Implantation	83	101	88	104
Mean No. Implants/Pregnancy	11.9	10.1	9.8	11.6
Pre natal Loss (%)	4.3	10.0	5.6	9.5
Post natal Loss (%)	0.0	1.3	0.0	0.0
Summary of Litter data
	Dosage levels mg/kg/day
	0	100	300	1000
Pairs started (no.)	10	10	10	10
Dams with live pup at birth (no.)	7	10	8	9
Dams with live pups at Day 4 pp (no.)	7	10	8	9
Live litter size at birth (mean)	11.3	9.00	10.1	10.7
Live litter size at Day 4 (mean)	11.3	9.00	9.6	10.7
Sex ratio (%) at birth (mean)	47.9	54.5	52.4	48.9
Sex ratio (%) at Day 4 (mean)	47.9	54.5	52.8	48.9
Litter weight at Day 1 (mean)	78.7	64.3	69.3	73.8
Litter weight at Day 4 (mean)	124.1	100.9	110.3	113.6
Pup weight at Day 1 (mean)	7.0	7.2	7.4	7.0
Pup weight at Day 4 (mean)	11.1	11.4	11.9	10.9
Pup weight at the Day of AGD (Day 1pp)
Mean males	7.30	7.30	7.30	7.30
Mean females	6.81	6.81	6.81	6.81
Pup AGD normalized (Day 1pp)
Mean males	2.23	2.23	2.23	2.23
Mean females	1.23	1.23	1.23	1.23
Male pup nipple present at Day 13 (no.)	0	0	0	0
Pup weight at Day 13 (mean)	31.8	32.6	33.5	31.2
Pups with major abnormalities at necopsy
Dams with 0	7	10	8	9
Dams with 1	0	0	0	0
Dams with > 2	0	0	0	0
Pre-natal loss (implantations minus live births)(no.)
Females with 0	4	6	5	4
Females with 1	2	1	2	3
Females with 2	1	1	1	1
Females with > 3	0	2	1	1
Post-natal (live births minus live at Day 1) (no.)
Females with 0	7	10	6	9
Females with 1	0	0	1	0
Females with 2	0	0	1	0
Females with > 3	0	0	0	0
Post-natal (live births at Day 4 after culling minus live Day 14) (no.)
Females with 0	7	9	8	9
Females with 1	0	1	0	0
Females with 2	0	0	0	0
Females with > 3	0	0	0	0

 

Conclusions:

The NOAEL for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.

Executive summary:

This screening study for reproductive and developmental toxicity was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats. 
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).

The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.

During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.

On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

EC Number:

226-970-7

EC Name:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

Cas Number:

5580-57-4

Molecular formula:

C43H35Cl5N8O6

IUPAC Name:

3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis[4-chloro-N-(3-chloro-2-methylphenyl)benzamide]

Test material form:

solid: nanoform

Details on test material:

Physical state/ appearance: yellow powder

Specific details on test material used for the study:

Batch no.: 0004460012
Purity: 99.3 wt%
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: Room temperature

The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to a quality system.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hannover

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Germany, D-97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8-10 weeks
- Weight at study initiation: (P) Males: 208-227 g; Females: 198 -217 g
- Housing: 5 of one sex to a cage until pairing; 1:1 (m/f) during pairing; after pairing: females individually, males as before mating
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum
- Acclimation period: 2 weeks

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

IN-LIFE DATES: From: 25 Nov 2021 To: Feb 2022 (females), Jan 2022 (males)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The preparations were made weekly and every three days at concentration of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
A 28 hour stability at room temperature and an 8 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in internal SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the formulations prepared on the first and the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in internal SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray
- After successful mating each pregnant female was caged: individually

Duration of treatment / exposure:

Males: 34/35 d
Females: 66 d

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), at least once daily (clinical signs)
- Cage side observations: mortality, clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: weekly before mating, on days 0, 7, 14 and 20 p.c.; on days 1, 4, 7 and 13 p.p. and prior necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food consumed by each cage and females was recorded weekly during the pre-mating period starting from the beginning of treatment up to mating. Individual food consumption for mated females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.

PARTUITION CHECK:
- three times a day during the working day and twice daily during the weekends and Public Holidays
- from Day 20 to Day 25 post coitum

OESTROUS CYCLE:
The assessment of oestrous cycles, by vaginal smears, was performed once daily in the morning in stock females and in allocated females for 2 weeks before the start of dosing.

IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH)

POSTMORTEM EXAMINATIONS:
- The clinical history of the males and females of the parental generation 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 and the required tissue samples preserved in fixative and processed for histopathological examination.
- All females were also examined for the number of visible implantation sites (pregnant animals). Uteri from females with no visible implantations, and uterus from one female with implantations only on the right horn, were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
- The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No

Blood sampling:

As part of the necropsy procedure, blood samples (approximately 0.8 mL) were withdrawn under isoflurane anaesthesia from the abdominal vena cava.

Fetal examinations:

STANDARTISATION OF LITTER:
- on Day 4 post partum,
- litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter
- Partial adjustment (for example, 5 males and 3 females) was acceptable
- no pups were eliminated when litter size dropped below the culling target (8 pups/litter).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths, live births
- weight gain (weighing on day 1, 4, 7, 13)
- postnatal mortality
- presence of gross anomalies
- anogenital distance (AGD)
- pup weight on the day of AGD
- presence of nipples/areolae in male pups (Day 13/14)

GROSS EXAMINATION OF DEAD PUPS

BLOOD COLLECTION:
- On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 mL (per sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).
In order to obtain the required volume, blood samples were pooled from two or three pups on Day 4 post partum.

IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH)

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.

Indices:

Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day when the parturition is defined complete (Day 0 post partum).

Males:

Copulatory Index (%) = no. of males with confirmed mating/no. of males cohabitated ×100

Fertility Index (%) = no. of males which induced pregnancy/no. of males cohabitated ×100

Females:

Copulatory Index (%) = no. of females with confirmed mating/no. of females cohabitated ×100

Fertility Index (%) = no. of pregnant females/no. of females cohabitated ×100

Pup loss at Day 0 post partum was calculated as a percentage from the formula:
(Total litter size−Live litter size)/Total litter size ×100

Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations−Live litter size at birth)/no. of visible implantations ×100

Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from
the formula:
(Live litter size at birth−live litter size at Day 4 (before culling))/Live litter size at birth ×100

Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from
the formula:
(Live litter size onDay 4 (after culling)−Live litter size onDay 13)/Live litter size onDay 4 (after culling) ×100

Males and females:

Pre coital Interval = The number of nights paired prior to the detection of mating

Historical control data:

T4 and TSH historical data for Wistar Hannover rat in serum were available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No differences in body weight were recorded between treated and control groups throughout the entire study.
A degree of variability in body weight gain was observed in both males and females over the course of the study and among all groups, but no relationship with the test item treatment was observed. Notably, there was a -58% decrease in body weight gain in high dose females at Day 7 post partum, compared to the control, but without statistical significance.
Being isolated event and without a dose-related trend, this case was not considered to be treatment related.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

effects observed, non-treatment-related

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

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

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significance decrease in mean thyroid weight was found in male pups of mid-dose group respect to than from control dams. This decrease (-21%) was slight and not dose related, therefore considered incidental. No other statistically significant changes were noted.

Effect levels (fetuses)

Key result

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

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 2: Reproduction data

Groups	1	2	3	4
Paired No.	10	10	10	10
Positive identification of mating No.	9	10	10	10
Not pregnant	3	0	1	1
Conceiving number	7	10	9	9
Total resorptions	0	0	1	0
Total litter loss	0	0	0	0
Positive identification of mating 1 - 5 days	9	9	8	7
Positive identification of mating 6 - 14 days	0	1	2	3
No. of females with live pups on Day 14 post partum	7	10	8	9

 

Table 3: Reproduction data and litter data - group means

Summary of Reproduction Data
	Dosage levels mg/kg/day
Observations	0	100	300	1000
No. Dams Inseminated	9	10	10	10
Oestrus cycle (mean number)	4	3	4	4
No. Dams Pregnant	7	10	9	9
Percent Dams Pregnant	70.0	100.0	90.0	90.0
Copulatory interval (1-5 days)	9	9	8	7
Copulatory interval (6-14 days)	0	1	2	3
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	1	0
No. of Litters	7	10	8	9
Total No. Implantation	83	101	88	104
Mean No. Implants/Pregnancy	11.9	10.1	9.8	11.6
Pre natal Loss (%)	4.3	10.0	5.6	9.5
Post natal Loss (%)	0.0	1.3	0.0	0.0
Summary of Litter data
	Dosage levels mg/kg/day
	0	100	300	1000
Pairs started (no.)	10	10	10	10
Dams with live pup at birth (no.)	7	10	8	9
Dams with live pups at Day 4 pp (no.)	7	10	8	9
Live litter size at birth (mean)	11.3	9.00	10.1	10.7
Live litter size at Day 4 (mean)	11.3	9.00	9.6	10.7
Sex ratio (%) at birth (mean)	47.9	54.5	52.4	48.9
Sex ratio (%) at Day 4 (mean)	47.9	54.5	52.8	48.9
Litter weight at Day 1 (mean)	78.7	64.3	69.3	73.8
Litter weight at Day 4 (mean)	124.1	100.9	110.3	113.6
Pup weight at Day 1 (mean)	7.0	7.2	7.4	7.0
Pup weight at Day 4 (mean)	11.1	11.4	11.9	10.9
Pup weight at the Day of AGD (Day 1pp)
Mean males	7.30	7.30	7.30	7.30
Mean females	6.81	6.81	6.81	6.81
Pup AGD normalized (Day 1pp)
Mean males	2.23	2.23	2.23	2.23
Mean females	1.23	1.23	1.23	1.23
Male pup nipple present at Day 13 (no.)	0	0	0	0
Pup weight at Day 13 (mean)	31.8	32.6	33.5	31.2
Pups with major abnormalities at necopsy
Dams with 0	7	10	8	9
Dams with 1	0	0	0	0
Dams with > 2	0	0	0	0
Pre-natal loss (implantations minus live births)(no.)
Females with 0	4	6	5	4
Females with 1	2	1	2	3
Females with 2	1	1	1	1
Females with > 3	0	2	1	1
Post-natal (live births minus live at Day 1) (no.)
Females with 0	7	10	6	9
Females with 1	0	0	1	0
Females with 2	0	0	1	0
Females with > 3	0	0	0	0
Post-natal (live births at Day 4 after culling minus live Day 14) (no.)
Females with 0	7	9	8	9
Females with 1	0	1	0	0
Females with 2	0	0	0	0
Females with > 3	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

The NOAEL for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.

Executive summary:

This screening study for reproductive and developmental toxicity was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats. 
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).

The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.

During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.

On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.