Ferrate(1-), bis[4-[2-[5-chloro-2-(hydroxy-.kappa.O)phenyl]diazenyl-.kappa.N1]-3-(hydroxy-.kappa.O)-N-phenyl-2-naphthalenecarboxamidato(2-)]-, ammonium (1:1)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Subacute NOEL (parental/offspring): 1000 mg/kg bw/day (OECD 421/GLP)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2011 - December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study performed in accordance to OECD Guideline 421

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: ~12 weeks
- Weight at study initiation: males weighed 307 to 351g, the females weighed 187 to 229g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 light / 12 dark

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Ltd. Project Number: 41100724). Results from the previous study showed the formulations to be stable for at least three hours. Formulations were therefore prepared daily.
Once weekly, samples of each test item formulation were taken and analysed for concentration of DL-N33 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 16. The results indicate that the prepared formulations were within ± 10% of the nominal concentration.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 6 ml/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The stability and homogeneity of the test item formulations were previously determined. Results from the previous study showed the formulations to be stable for at least three hours.
- Amount of vehicle (if gavage): 6ml

Details on mating procedure:

- M/F ratio per cage:On Day 15, animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: a maximum of fourteen days.
- Proof of pregnancy:Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day0 of gestation)
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of DL-N33 in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test item formulations were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 0.02 mg/ml. Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.02 mg/ml. The standard and sample solutions were analysed by HPLC. Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test item.
All results were within ±10% of nominal concentration values.

Frequency of treatment:

daily

Details on study schedule:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) On Day 15, animals were paired on a 1 male: 1 female basis within each dosegroup for a maximum of fourteen days.

iii) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.

iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post parium. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

v) The male dose groups were killed and examined macroscopically on Day 43.

vi) At Day 5 post parium, all females and surviving offspring were killed and examined macroscopically.

Remarks:

Doses / Concentrations:
5 mg/ml
Basis:
nominal conc.
30 mg/kg bw/day

Remarks:

Doses / Concentrations:
50 mg/ml
Basis:
nominal conc.
300 mg/kg bw/day

Remarks:

Doses / Concentrations:
167 mg/ml
Basis:
nominal conc.
1000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Other:

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes / No / No data
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post parium.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4). Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water intake was observed daily by visual inspection of water bottles for any overt changes.
Intergroup differences did not indicate any need for more formal gravimetric measurements.

OTHER:

Litter observations:

On completion of parturition (Day 0 post parium), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post parium
iii) Sex of offspring on Days 1 and 4 post parium
iv) Clinical condition of offspring from birth to Day 5 post parium
v) Individual offspring weights on Days 1 and 4 post parium (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post parium.

Postmortem examinations (parental animals):

Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post parium. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (offspring):

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Statistical Analysis
The following parameters were subjected to statistical analysis:
Body weight and body weight change
Food consumption for females during gestation and lactation
Pre-coital interval and gestation length
Litter size and litter weights
Sex ratio
Corpora lutea and implantation sites
Implantation losses and viability indices
Offspring body weight and body weight change
Offspring surface righting
Adult absolute and body weight-relative organ weights (Males)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinically observable signs of toxicity were detected. Clinical signs were confined to the presence of black coloured staining of the fur by the test item formulations and post-dose increased salivation for animals of either sex treated with 1000 or 300 mg/kg bw/day. Incidences of increased salivation were also evident for two males treated with 30 mg/kg bw/day. Increased salivation is commonly observed following the oral administration of a slightly irritant or unpalatable test item formation, and in the absence of any supporting data to suggest irritancy, the clinical signs observed in this study were considered to be unrelated to systemic toxicity.

BODY WEIGHT
No adverse effects on body weight change were detected for treated males when compared to controls.
Males treated with 1000 mg/kg bw/day showed slight but statistically significant reductions in body weight gains when compared to control values during Weeks 3, 5 and 6. The significances achieved were minimal (P<0.05), and in isolation, these slight reductions were not considered to represent an adverse effect of treatment.
No adverse effects on body weight change were evident for females during the premating, gestation or lactation phases of the study.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effects on dietary intake or food conversion efficiency were detected. Slightly higher dietary intakes were noted for males treated with 1000 mg/kg bw/day in comparison to controls (approximately 5-10% higher). Slightly lower food efficiency values were also evident for males treated with 1000 mg/kg bw/day during Weeks 5 and 6. Increases in dietary intake were also evident for males treated with 300 mg/kg bw/day, although a convincing dose-related response was not evident. In the absence of any adverse effects on general health or reproductive performance, these differences were considered not to represent an adverse effect of treatment.
Females treated with 1000 mg/kg/day showed increases in dietary intake during the premating phase of the study when compared to controls (approximately 11 %). Statistically significant increases in dietary intake were evident for this group during the gestation phase of the study when compared to control values (P<0.05 - P<0.01). Dietary intake during early lactation for high dose females was comparable to controls. No such effects were evident for females treated with 300 and 30 mg/kg bw/day. In the absence of any adverse effects on reproduction, these increases were considered not to represent an adverse effect of treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating
No treatment-related effects were detected in mating performance. The majority of animals mated within four days of pairing. Statistical analysis of the pre-coital data did not reveal significant intergroup differences.
Fertility
No treatment~related effects on fertility were detected. All females achieved pregnancy; however one female from the low dose group did not produce a live litter.
Gestation Length
No treatment-related effects were detected in the length of gestation between control and treated groups. The majority of females showed a gestation length between 22% and 23% days. Statistical analysis of the gestation length data did not reveal any significant intergroup differences.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Slightly elevated absolute and body weight-relative testes weights were evident for males treated with 1000 mg/kg bw/day when compared to controls. The significance achieved was minimal (P<0.05), and two males showed values outside of the normal expected ranges. However, in the absence of any histopathological correlates or adverse effects on reproduction, this finding was considered to have arisen incidentally and did not represent a true effect of treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Offspring
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Adults
No treatment-related macroscopic abnormalities were detected in the tissues examined from the adults.
Black coloured contents were evident in the GI tract for animals of either sex treated with 1000 mg/kg bw/day and for males treated with 300 mg/kg bw/day. This was attributable to the coloured test item and was of no toxicological importance.
A small prostate and seminal vesicles were recorded for one control male however this did not impact reproductive performance.
One female treated with 30 mg/kg bw/day was terminated after Day 25 post coitum following the absence of a litter; however one foetus was found in utero confirming the pregnancy status of this female.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of reproductive tissues from the high dose group did not reveal any treatment-related changes.
All microscopic findings recorded were considered to be incidental in nature.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
No significant differences in the number or corpora lutea or implantation sites were noted for treated females when compared to controls. The percentage of pre- and postimplantation losses for treated females was comparable to controls.
No differences in sex ratio or litter size were evident for offspring from treated litters when compared to those from the controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No differences in litter weights were evident in the treated groups when compared to controls.
No clinically observable signs of toxicity were detected. Isolated instances of small size, offspring found dead or missing from the litter are commonly observed findings in studies of this type and considered to be unrelated to test item toxicity. Two of the offspring found dead showed autolytic changes. The remaining interim death offspring, and all remaining offspring killed at study termination did not show any macroscopic abnormalities.
Statistical analysis of the data revealed no significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

The oral administration of DL-N33 to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any treatment-related effects in both parents or offspring, therefore a clear 'No Observed Effect Level' (NOEL) was established at 1000 mg/kg bw/day for reproductive toxicity.

Executive summary:

Introduction

The study was performed to screen for potential adverse effects of the test item on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study was designed to be compatible with the DECD Guidelines for Testing of Chemicals No. 421 "Reproduction/Developmental Toxicity Screening Test" (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHanTM:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post parium. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results

Adult Responses:

Mortality:There were no unscheduled deaths. Clinical Observations: No toxicologically significant clinical observations were detected. Body Weight: No adverse effects on body weight change were evident for males during the treatment period or for females during the pre-mating, gestation and lactation phases of the study when compared to controls. Food Consumption and Food Efficiency: No adverse effects on dietary intake or food conversion efficiency were detected for treated males when compared to controls during treatment, or for treated females during the pre-mating, gestation or lactation phases of the study, in comparison to controls. Water Consumption. Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake. Reproductive Screening: Mating. No treatment-related effects were detected in mating performance. Fertility. No treatment-related effects on fertility were detected. All females achieved pregnancy. Gestation Lengths. No treatment-related effects were detected in the length of gestation between control and treated groups. Litter Responses: Offspring Litter Size, Sex Ratio and Viability. No differences in sex ratio or litter size were evident for offspring from treated litters when compared to those from the controls. Offspring Growth and Development. No differences in litter weights were evident in the treated groups when compared to controls. There were no clinically observable signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill. Uterine Examinations. No significant differences in the number or corpora lutea or implantation sites were noted for treated females when compared to controls. The percentage of pre- and post-implantation losses for treated females were comparable to controls. Pathology: Necropsy. No treatment-related macroscopic abnormalities were detected. Organ Weights. No toxicologically important organ weight changes were detected. Histopathology. No treatment-related microscopic changes were detected.

Conclusion.

The oral administration of DL-N33 to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, did not result in any treatment-related effects in both parents or offspring, therefore a clear 'No Observed Effect Level' (NOEL) was established at 1000 mg/kg bw/day for reproductive toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is one study available and it is OECD/GLP compliant.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is one reproduction/developmental toxicity screening test in rats available.

In a reproduction/developmental toxicity screening test (OECD 421/GLP), DL-N33 was administered to 4 groups of Wistar Han:RccHanTM:WIST rats (10 animals/sex/group) by gavage in arachis oil at dose levels of 0, 30, 300 and 1000 mg/kg bw/day, 7 days per week, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females).

The analytics indicated that the prepared formulations were within ± 10% of the nominal concentration.

There were no unscheduled deaths. No toxicologically significant clinical observations were detected. No adverse effects on body weight change were evident for males during the treatment period or for females during the pre-mating, gestation and lactation phases of the study when compared to controls. No adverse effects on dietary intake or food conversion efficiency were detected for treated males when compared to controls during treatment, or for treated females during the pre-mating, gestation or lactation phases of the study, in comparison to controls.

No treatment-related effects were detected in mating performance. No treatment-related effects on fertility were detected. All females achieved pregnancy. No treatment-related effects were detected in the length of gestation between control and treated groups. No differences in sex ratio or litter size were evident for offspring from treated litters when compared to those from the controls. No differences in litter weights were evident in the treated groups when compared to controls. There were no clinically observable signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill. No significant differences in the number or corpora lutea or implantation sites were noted for treated females when compared to controls. The percentage of pre- and post-implantation losses for treated females were comparable to controls. No treatment-related macroscopic abnormalities were detected upon necropsy. No toxicologically important organ weight changes were detected. No treatment-related microscopic changes were detected.

The NOEL (male/female) was 1000 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information in the dossier, the conclusion for the substance DL-N33 (CAS No. 104815 -18 -1) does not need to be classified for reproductive toxicity when the criteria outlined in Annex I of 1227/2008/EC are applied.