Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 April 2016 to 16 Jan 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008

Version / remarks:

30 May 2008 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).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals: 15 days
- Basis for dose level selection: Dose levels were selected in collaboration with the sponsor based an available toxicological data, including a Rat Oral Gavage Fourteen Day Range Finding Toxicity Study performed at this Test Facility (Study Number WP89QB), where a dosage of 1000 mg/kg bw/day AI was considered to be well tolerated. Dosages of 0 (Control), 100, 300 and 1000 mg/kg bw/day AI were therefore selected for use on this study.
- Inclusion/exclusion of extension of Cohort 1B: 14 Days
- Route of administration: Oral Gavage

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

279-365-5

EC Name:

Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

80010-51-1

Molecular formula:

C29H28Cl2N4O7S.Na

IUPAC Name:

sodium 1-amino-4-[(3,5-bis{[(chloroacetyl)amino]methyl}-2,4,6-trimethylphenyl)amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: POLAR BLAU RLS (FAT 21'036/D)
Description: solid
EN-Number: 384481.22
Purity / formulation: approx. 87% (a.i)
Stability of test article: Stable at storage conditions
Expiration date: 04/99
Stability of test article in vehicle: unknown in vaselinum album, excluded from the Statement of Compliance
Storage conditions: at room temperature

Specific details on test material used for the study:

Identification: FAT 21036/G TE
Physical State/Appearance: Blue solid
Chemical Name: Sodium 1-amino-4-[[(3,5-bis[[(chloroacetyl)amino]nethyl]-2,4,6-trimethylphenyl]amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
Purity: 63.5 %
Batch Number: AT-0033945200
Date Received: 14 March 2016
Storage Conditions: Ambient temperature in the dark
Expiry Date: 13 April 2020

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST strain

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 12 wks;
- Weight at study initiation: (P) Males: 317-357 g; Females:190-231 g
- Fasting period before study: none
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing 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.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ”C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 June 2016 To: 25 July 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least eleven days when stored at 4 °C in the dark. Formulations were therefore prepared on a weekly basis and stored at approximately 4 °C in the dark.

GROUP Adjusted for Purity Test Item as supplied
Dose level Concentration Treatment vol Concentration (mg/ml)
(mg/kg bw/day) (mg/ml) (ml/kg)
Control 0* 0* 20 0*
Low 100 5 20 7.87
Imtermediate 300 15 20 23.6
HIgh 1000 50 20 78.7

* Control animals were treated with vehicle (distilled water) alone.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to asday 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLCUV) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

HPLC conditions:
HPLC: Aglient Technologies 1200
Column: Gemini 3µ C18 (100 x 4.6mm id) at 30 ”C
Mobile Phase: Eluent A : water/TBAB solution (90:10 v/v)
Eluent B: acetonitrile/TBAB solution (90:10 v/v)
TBAB solution: 5g Tetrabutylammonium bromide in 250 mL acetonitirile
Gradient: Time % B
0 0
1 0
6 100
11 100
11.5 0
15 0
Flow rate: 1 mL/min
UV: 420 nm
Injection volume: 25 μL
Retention time: 5.5 mins

Linearity
Calibration data was found to be linear over the range 0.00508 to 0.1524 mg/mL

Method accuracy and precison
Mean recovery (accuracy) and precision at 2 mg/mL = 99 % (CV=1.23 %; n=5)
Mean recovery (accuracy) and precision at 320 mg/mL = 102 % (CV=0.348 %; n=5)
LOQ (defined aslowest standard concentration) = 0.00508 mg/mL

Test item analysis
The mean concnetrations fo test item in the test formulations were within ±10 % nominal concentrations

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

Daily
To reduce the viscosity of the dosing formulations a dosage volume of 20 mL/kg bw was utilized for dosing in this study. In order to reduce the potential impact of this increase in dosage volume on the animals, dosing on this study on each day was spilt over two occasions approximately four hours apart.

Details on study schedule:

Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated twice 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 dose group 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 partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Guideline requirement
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Individual clinical observations were performed immediately before dosing, up to thirty minutes post dosing and one hour after each dosing occasion. All observations were recorded. Additional observations may be included at the discretion of the Study Director or for the monitoring of animal health.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE
During the pre-pairing 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-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes

Litter observations:

Litter Data
On completion of parturition (Day 0 post partum), 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 partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development: All live offspring were assessed for surface righting reflex on Day 1 post partum.

Postmortem examinations (parental animals):

Terminal Investigations
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 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). The corpora lutea were also counted.
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.

Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland Prostate
Epididymides Seminal vesicles
Ovaries Testes
Mammary gland (females only) Uterus/Cervix
Pituitary Vagina

The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination were not extended to include animals in the low and intermediate groups.

Statistics:

Statistical Analysis
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Reproductive indices:

The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number mater/number paired x 100
Pregnancy Index (%) = Number of pregnant females/number mated x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = No.females delivering live offspring/no. pregnant females x 100

Offspring viability indices:

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = No. offspring alive on Day 1/No. offspring born x 100
Viability Index (%) = No. offspring alive on Day 4/No. offspring alive on Day 1 x 100

iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment. Female number 65 was found dead on Day 6. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue colored liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body (including the heart, kidneys, liver, lymph nodes, mammary gland, muscle, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract).

Male number 55 was found dead on Day 18. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue coloured liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body, including the adrenals, heart, bone marrow, kidneys, liver, lymph nodes, mammary gland, muscle, oesophagus, pancreas, seminal vesicles, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

For males at 1000 mg/kg bw/day, there was a suggestion of slightly lower mean body weight gain during the first week of treatment, compared to control. However, there was no statistical significance and, while three males showed mean body weight loss during this period, body weight gains for the remaining animals were within the concurrent control range. Subsequent mean body weight gains of males (with the exception of Days 29 to 36) were generally similar to, but slightly lower than, control, with differences failing to attain statistical significance. Overall mean body weight gain at the end of the study was also slightly lower than control but again there was no statistical significance. There was no obvious effect of treatment on body weight and body weight gain of males at 100 or 300 mg/kg bw/day. At all dosages higher mean body weight gain for males during Week 5 (Days 29 to 36) attained statistical significance when compared with control. However there was no dosage relationship and this isolated increase was considered to be incidental and unrelated to treatment. There was no obvious effect of treatment on body weight and body weight gain of females during the two week pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no obvious effect of treatment on food consumption for males during the prepairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. There was no obvious consistent effect of treatment on food conversion efficiency although values for males at 1000 mg/kg bw/day were slightly lower than control during the first week of treatment.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Several animals were not pregnant following mating. These occurred across all Groups including controls and no findings were noted in these animals or their corresponding male partners to account for the lack of pregnancy. Animal 38 (Group 2 female) appeared to have been pregnant and was lactating. There was evidence of uterine infection but no indication of an effect due to the administration of the test item. There were no treatment-related pathological findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. Female 65 (Group 3) was found dead early in the study. There were no findings at histopathology to account for death. Male 55 (Group 3) was also found dead without significant histopathological change.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating
There was no obvious effect of treatment on mating, as assessed by pre-coital interval, at 100, 300 or 1000 mg/kg bw/day. At 100 mg/kg bw/day one male/female pairing failed to mate, but this isolated occurrence, in the absence of any similar occurrence at 1000 mg/kg bw/day, was considered to be incidental and unrelated to treatment.

Fertility
There was no obvious effect of treatment on fertility, as assessed by pregnancy rate, at 100, 300 or 1000 mg/kg bw/day.
A total of 2, 1, 0 and 1 females at 0 (control), 100, 300 or 1000 mg/kg bw/day failed to achieve pregnancy. The incidence of non-pregnancy showed no dosage relationship and was considered to be unrelated to treatment.

Gestation Length
The intergroup distribution of gestation lengths did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Litter responses
At 1000 mg/kg bw/day, one female achieved pregnancy but was not observed to give birth to a litter; therefore this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. The female only had three implantations and it is not unusual for such small litters not to be maintained to term; in isolation this finding is considered to be unrelated to maternal treatment.
For one female at 100 mg/kg bw/day, no implantations were apparent at macroscopic necropsy but microscopic evaluation of the uterus indicated that the female had been pregnant. Again this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. this isolated occurrence was considered to be unrelated to treatment.
Additionally, as previously indicated, a total of 2, 1, 0 and 1 mated females failed to achieve pregnancy in the 0 (control), 100, 300 and 1000 mg/kg bw/day groups respectively. The following assessment is based on the 10, 9, 11 and 10 females that successfully maintained a litter to Day 4 of lactation.

Offspring Litter Size, Sex Ratio and Viability
There was considered to be no effect of treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects seen

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The low incidence of clinical sign observed for the offspring did not indicate any obvious underlying effect on offspring growth and development at any of the dosages investigated.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was considered to be no obvious effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 at 100, 300 or 1000 mg/kg bw/day.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects seen

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

no

Any other information on results incl. tables

Analytical investigation: The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity and stability was confirmed for test item in water formulations at nominal concentrations of 7.87 mg/mL and 78.7 mg/mL when stored refrigerated for 11 days. The mean concentratio of test item waas within 10% of nominal concentratios with the exception of analysis 2, comfirming accurate formulation.

Applicant's summary and conclusion

Conclusions:

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.

Executive summary:

A 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 is compatible with the requirements of the recommendations of the OECD 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).

Method: The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 63.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) over the same treatment period. 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. Surviving adult males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results

Adult Responses

Mortality

There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment.

Clinical Observations

There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day. Fur staining by the Test Item was apparent at 1000 mg/kg bw/day and to a lesser extent at 300 mg/kg bw/day. One female at 100 mg/kg bw/day also showed similar fur staining on Day 6 of the study. Staining of the fur was not unexpected given the colored nature of the test item.

Body Weight

There was no obvious adverse effect of treatment on body weight or body weight gain for males during the pre-pairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.

Food Consumption

There was no obvious effect of treatment on food consumption for males during the prepairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.

There was no obvious adverse effect of treatment on food conversion efficiency for either sex during the pre-pairing phase of the study or for males during post-pairing study phase at 100, 300 or 1000 mg/kg bw/day.

Water Consumption

Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.

Reproductive Performance

Mating: There was no obvious effect of treatment on mating, as assessed by pre-coital interval, at 100, 300 or 1000 mg/kg bw/day.

Fertility: There was no obvious effect of treatment on fertility, as assessed by pregnancy rate, at 100, 300 or 1000 mg/kg bw/day.

Gestation Length: The intergroup distribution of gestation lengths did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: There was considered to be no effect of maternal treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and Day 4 and offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day.

Offspring Growth and Development

There was considered to be no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.

Offspring Observations

The low incidence of clinical sign observed for the offspring did not indicate any obvious underlying effect on offspring growth and development at any of the dosages investigated.

Necropsy

Offspring :Necropsy findings did not indicate any obvious effect of maternal treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Adults: Macroscopic necropsy revealed blue coloration or blue contents for various regions of the gastrointestinal tract for both sexes at 1000 mg/kg bw/day. A lower incidence of similar findings were apparent for both sexes at 100 and 300 mg/kg bw/day. This finding was consistent with the colored nature of the test item.

Organ Weights: There was no obvious effect of treatment on absolute or body weight relative testes or epididymides weights at 100, 300 or 1000 mg/kg bw/day.

Histopathology: Microscopic evaluation of male reproductive tissues at 1000 mg/kg bw/day did not indicate any obvious effect of treatment.

Conclusion

Based on the results of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.