Reaction mass of lithium sodium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxynaphthalene-2,7-disulfonate 2,2'-(methylimino)diethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There is no specific study on fertility assessment available.
For Bayscript Blaukomponente a 28-day study is available (Schladt 2013). During the 28-day treatment periodmale and female Wistar rats received the test substance by gavage 0, 100, 300 and 1000 mg/kg bw/day The study was performed according to OECD TG 407 and GLP. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day (limit dose).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

other justification

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Reproductive effects observed:

not specified

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:

The available subacute oral study is performed according to OECD TG 407 and GLP and is evaluated with Klimisch score 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Studies dealing specifically with toxicity to reproduction (fertility assessment) of Bayscript Blaukomponente are not identified. In addition, there is no screening study according to OECD TG 421 available. However there is a Repeated Dose Toxicity Study available in rats over a period of 28 days in which rats were dosed up to and including 1000 mg/kg bw/day.

As confirmed by literature (Mangelsdorf et al 2003, Ulbrich & Palmer 1995, Janer et al 2007a, Dent 2007) histopathological examinations in repeated dose toxicity are of high value and high sensitivity for evaluation of reproductive toxicity. Therefore, at least for fertility assessment, repeated dose toxicity study showing no adverse effect should be taken into account. Also it is agreed that histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. Therefore repeated dose toxicity studies should be considered sufficient information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered by the repeated dose toxicity study and the mode of action of the test substance does not give evidence for a specific toxicity (as would e.g. sex hormone receptor binding activity).

Bayscript Blaukomponente (konserviert) was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks. This GLP study was performed according to OECD guideline 407.

Treatment with Bayscript Blaukomponente resulted in several findings which were related to the color of the test substance. The whole body and several organs of animals treated with 1000 mg/kg bw/day appeared discolored. The kidneys were also discolored in the lower dose groups.

In the kidneys, beneath findings associated with the color of the test substance (starting at 300 mg/kg b.w./day), corticotubular degeneration and increased incidence and/or severity of cortical basophilic (regenerative) tubules were noted at 1000 mg/kg b.w./day. Furthermore, demyelinated fibers in nerve roots at the spinal cord were seen in both sexes at 1000 mg/kg b.w./day.

At 1000 mg/kg b.w./day, histopathological findings in the mesenteric lymph node, the interstitium of the testis, and in the Peyer's patch were related to the colored test item and not accompanied by other structural changes of the organs.

Minor changes in red blood cell parameters observed for males and females at 1000 mg/kg b.w./day were associated with appearing of golden-brown pigment deposition in the spleen in females. In males, the thrombocyte count was increased at 1000 mg/kg b.w./day.

Despite this general toxic effects no pathological changes were observed at the reproductive organs in male and female rats

Under the conditions described the no adverse observed effect level (NOAEL) for administration of BAYSCRIPT Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to mild to moderate effects on kidneys and minor changes in nerve roots at 1000 mg/kg bw/day.

With respect to reproductive organ toxicity a NOAEL of 1000 mg/kgbw/day is established for male and female rats.

In conclusion, due to the lack of adverse effects on reproductive organs up to and including the limit dose of 1000 mg/kg bw/day over 28 days, there is no indication of a specific toxic potential of Bayscript Blaukomponente to the reproductive organs. Therefore, based on the considerations above, no further testing should be required for fertility assessment.

This is in accordance to ANNEX IX Section 8.7 column 2 of Regulation (EC) No 1907/2006 (REACH): Reproductive studies need not to be conducted if the substance is of low toxicological activity. Bayscript Blaukomponente is practically non-toxic following acute oral and dermal application (LD50 > 2000 mg/kg bw), is not irritating to the skin and eyes, does not show sensitizing potential and developed no mutagenic activity in the in-vitro tests (MNT, HPRT and Ames test) and a NOAEL of 300 mg/kg bw/day in male and female rats is the result in a subacute oral study based on changes only at the highest test dose of 1000 mg/kg bw/day (limit dose).

Overall, based on the lack of effects on reproductive organs in the subacute toxicity study there is no evidence that reproductive toxicity might occur. A developmental study according to OECD TG 414 was conducted and the requirements of the REACH Regulation for this tonnage band are fulfilled.

References

Mangelsdorf. et al., 2003: Some aspects relating to the evaluation of the effects of chemicals on male fertility. Regulatory toxicology and Pharmacology 36, 69-98

Ulbrich & Palmer, 1995: Detection of effects on male reproduction – a literature survey. J am. College of Toxicology 14, 293-327

Janer et al., 2007: A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study. Reproductive Toxicology 24, 103-113

Dent, 2007: Strength and limitations of using repeated-dose toxicity studies to predict effects on fertility. Regulatory Toxicology and Pharmacology 48, 241-258

Short description of key information:
There is no specific study on fertility assessment available.
For Bayscript Blaukomponente a 28-day study is available (Schladt 2013). During the 28-day treatment periodmale and female Wistar rats received the test substance by gavage 0, 100, 300 and 1000 mg/kg bw/day The study was performed according to OECD TG 407 and GLP. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day (limit dose).

Justification for selection of Effect on fertility via oral route:
There is no specific study on fertility assessment available.
For Bayscript Blaukomponente a 28-day study is available (Schladt 2013). During the 28-day treatment periodmale and female Wistar rats received the test substance by gavage 0, 100, 300 and 1000 mg/kg bw/day. The study was performed according to OECD TG 407 and GLP. Reproductive organs of all rats were weighed and the reproductive organs of the highest dose group and of the control animals were examined histopathologically. No adverse effects were noted from these organs in these groups. Based on these results there are no indications for specific adverse effects on the reproductive organs up to and including 1000 mg/kg bw/day (limit dose).

Effects on developmental toxicity

Description of key information

In an OECD 414 study Bayscript Blaukomponente MDA was administered to rats by oral gavage during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day. Bayscript Blaukomponente MDA produced no maternal toxicity or any effects on embryofetal survival or development.

The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

The influence of Bayscript Blaukomponente MDA on embryo-fetal survival and development was assessed in the Han Wistar rat, when administered during the organogenesis and fetal growth phases of pregnancy (Days 6-19 after mating).
Three groups of 22 females received Bayscript Blaukomponente MDA at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, purified water, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, kidney weights and the gravid uterus weights were recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination, including cartilaginous examination.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animal Information
Animals
Strain/Species: RccHan™;WIST rat.
Supplier: Envigo RMS Limited.
Number of animals ordered: 96 time mated females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimation: 5 days before commencement of pairing.
Age of the animals at the
start of the study (Day 1 of
gestation): 11 to 12 weeks old.
Weight range of the animals
at the start of the study
(Day 1 of gestation): 178 to 241 g

Allocation and Identification
Allocation: On arrival (Day 1 of gestation).
Method: Allocation was controlled to prevent any mating male from providing more than one mated female in each treatment group.
Identification of animals: Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.

Animal Care and Husbandry
Environmental Control
Rodent facility: Limited access - to limit entry of external biological and chemical agents and to limit the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
Lighting: Artificial lighting, 12 hours light : 12 hours dark with simulated periods of dawn and dusk..
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid (Tecniplast, UK); changed at appropriate intervals.
Cage distribution: The cages constituting each group were blocked by group and mounted in batteries.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding (JRS Lignocel, Mansfield, UK), which was changed at appropriate intervals each week.
Number of animals per cage: One female.

Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet (SDS, Special Diet Services, Witham, UK). The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed three times per week.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Formulation
Vehicle: Purified water.
Validated concentration range: 1 mg/mL to 100 mg/mL.
Method of preparation: A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test item formulation supplied.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8°C).
Test item accounting: Detailed records of compound usage were maintained.

Formulation Analysis
Stability: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability of the test item in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 2 of treatment were analyzed for achieved concentration of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item: Bayscript Blaukomponente MDA
EC number: EC 916-916-7
Appearance: Dark blue liquid
Purity: 30.8% (w/w) dyestuff
Purity/weighing factor: The test item is a reaction mass which cannot be separated from water therefore a correction factor was not applied.

Details on mating procedure:

Time mated females were used.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Duration of test:

Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Dose / conc.:

100 mg/kg bw/day

Remarks:

Bayscript Blaukomponente MDA

Dose / conc.:

330 mg/kg bw/day

Remarks:

Bayscript Blaukomponente MDA

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Bayscript Blaukomponente MDA

No. of animals per sex per dose:

22 female animals per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels selected for this study are 100, 330 and 1000 mg/kg/day as supplied (active material: 30.8, 102 and 308 mg/kg/day).
In a preliminary rat embryo-fetal survival and development study, administration of Bayscript Blaukomponente MDA to rats by oral gavage during the organogenesis phase of pregnancy (Days 6 to 19 after mating) at 250, 500 and 1000 mg/kg/day (active material: 77, 154 and 308 mg/kg/day) was well tolerated. The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day, therefore target doses of 100, 330 or 1000 mg/kg/day were considered acceptable for this study.

Maternal examinations:

Serial Observations

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimation period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily at the following times in relation to dose administration:
Predose observation
As each animal returned to its home cage 1 to 2 hours after completion of dosing As late as possible in the working day

Clinical Signs
A detailed physical examination was performed on each animal on Days 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 1, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 1-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations

Necropsy
All adult females were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Examination included a detailed assessment and documentation of any color changes in the internal organs, adipose tissue or skin. If color changes were observed, representative photographs were taken before retained tissues were placed in fixative.
Schedule Females surviving until the end of the scheduled study period were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison to minimise effects from the timing of the necropsy across groups.

Organ weights
Kidneys were weighed for adult females.

Ovaries and uterine content:

Reproductive Assessment
For all females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals:
For each ovary/uterine horn the number of: Corpora lutea, Implantation sites, Resorption sites (classified as early or late), Fetuses (live and dead).
Apparently non-pregnant animals and for apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide.

Fetal examinations:

Fetal Examination and Processing
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter. Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and stained with Alizarin Red and Alcian blue.

Fetal Pathology Examination:
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities
Alizarin Red and Alcian blue stained fetuses: Assessed for skeletal and cartilage development and abnormalities.

Statistics:

A statistical analysis was performed.

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations): (Number of corpora lutea)] : x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = [(Number of implantations – Number of live fetuses): (Number of implantations)] x 100

All group values and SD (as appropriate) were calculated from the individual litter values.

Historical control data:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan™;WIST strain was used because of the availability of historical control data.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs associated with dosing observed, and there were no test item related clinical signs during the detailed weekly physical examination.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There were no mortalities.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects on bodyweight or gravid uterine weights.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item related effects on food consumption.

Food efficiency:

no effects observed

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

not specified

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

Description (incidence and severity):

There were no test item related effects on body weight adjusted kidney weights on Day 20 of gestation

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopically, the kidneys of the dams given 330 or 1000 mg/kg/day were dark. This finding was considered to be related to the colour of the test item and therefore considered not to be adverse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day produced no maternal toxicity.

Number of abortions:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Other effects:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Details on maternal toxic effects:

Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day produced no maternal toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day produced no maternal toxicity.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Group mean placental, litter and fetal weights were similar in all groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item related effects on any gestation or litter parameters evaluated (implantation rate, pre and post implantation loss, litter size and sex ratio).

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There was no adverse effect of maternal treatment on the incidence of major and minor abnormalities and skeletal variants.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There was no adverse effect of maternal treatment on the incidence of major and minor abnormalities and skeletal variants.

Visceral malformations:

no effects observed

Description (incidence and severity):

There was no adverse effect of maternal treatment on the incidence of major and minor abnormalities and skeletal variants.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at100, 330 or 1000 mg/kg/day produced no maternal toxicity or any effects on embryofetal survival or development.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remarks'

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Litter data - group mean values on Day 20 of gestation

Dose Group Dose (mg/kg/day)	Control 1 0	MDA 2 100	MDA 3 330	MDA 4 1000
Group/ Sex		Corpora Lutea	Implantations	Resorptions Early Late Total			Live Young Male Female Total			Sex ratio (%M)	Implantation Loss (%) Pre- Post-
1F	Mean SD N	11.7 1.68 21	10.4 2.38 21	0.9 21	0.0 21	0.9 21	5.0 2.12 21	4.5 2.02 21	9.5 2.60 21	53.3 21	1.4 21	9.4 21
2F	Mean SD N	12.4 1.42 17	10.9 2.29 17	0.8 17	0.0 17	0.8 17	4.6 2.00 17	5.5 2.03 17	10.1 2.47 17	45.9 17	12.6 17	8.3 17
3F	Mean SD N	11.4 1.70 20	10.5 2.16 20	0.7 20	0.1 20	0.8 20	4.9 1.90 20	4.9 1.87 20	9.7 2.36 20	50.5 20	8.9 20	7.2 20
4F	Mean SD N	12.0 2.46 21	10.1 2.91 21	0.5 21	0.0 21	0.6 21	4.1 1.80 21	5.4 2.46 21	9.5 3.01 21	46.1 21	17.9 21	6.3 21

Fetal examinations - major abnormality findings - group incidences

Dose Group Dose (mg/kg/day)	Control 1 0	MDA 2 100	MDA 3 330	MDA 4 1000
		Placental Weight	Litter Weight	Litter Size	Male Fetal Weight		Female Fetal Weight		Overall Fetal Weight
1F	Mean SD N	0.52 0.048 21	34.96 9.329 21	9.48 2.600 21	3.79 0.269 21		3.63 0.200 21		3.71 0.223 21
2F	Mean SD N	0.53 0.089 17	37.77 9.604 17	10.12 2.472 17	3.83 0.264 17		3.68 0.321 17		3.75 0.290 17
3F	Mean SD N	0.53 0.060 20	36.20 8.420 20	9.70 2.364 20	3.82 0.245 20		3.68 0.195 20		3.75 0.213 20
4F	Mean SD N	0.55 0.149 21	35.84 11.588 21	9.52 3 .010 21	3.92 0.311 21		3.68 0.284 20		3.80 0.340 21

Litter data - group mean values on Day 20 of gestation

Dose Group Dose (mg/kg/day)	Control 1 0	MDA 2 100	MDA 3 330	MDA 4 1000
		Fetuses					Litters
Group Number examined total Number Affected		1 199 1	2* 172 1	3 194 1	4 200 2		1 21 2		2* 17 1				3 20   1	4 21   2
Head Visceral	Microphthalmia/absent optic nerve	0	1	0	0		0		1				0	0
Cervical/Thoracic Visceral	Dorsally displaced pulmonary trunk Muscular ventricular septal defect Diaphragmatic hernia	0 0 1	0 0 0	0 0 1	1 1 0		0 0 1		0 0 0				0 0 1	1 1 0
Lumbar (and abdominal)/Sacral/Caudal Visceral	Imperforate anus Anury	0 0	0 0	0 0	1 1		0 0		0 0				0 0	1 1

Note: Individual fetuses/litters may occur in more than one category.

* Litter 28 excluded from group incidences – Suspected Day 21

Fetal examinations - minor skeletal abnormality and variants findings - group incidences

Dose Group Dose (mg/kg/day)	Control 1 0	MDA 2 100	MDA 3 330	MDA 4 1000
		Fetuses					Litters
Group Number examined		1 100	2* 85	3 99	4 99		1 21		2* 17				3 20	4 21
Minor skeletal abnormalities Ribs Sternebrae Costal cartilage Total affected by one or more of the above	medially thickened/kinked bipartite ossified misaligned ossification sites misaligned hemicentres misshapen branched 1st not connected to sternum partially fused misaligned interrupted 7th not connected to sternum hole in xiphoid branched xiphoid Total affected by one or more of the above	0 1 2 1 0 0 0 0 1 7 1 4 1 15	1 0 2 0 0 1 1 1 0 2 0 1 1 8	0 0 1 0 0 0 0 1 0 6 1 4 2 13	0 0 0 1 1 0 0 0 1 1 0 4 0 7		0 1 2 1 0 0 0 0 1 7 1 3 1 12		1 0 2 0 0 1 1 1 0 2 0 1 1 7				0 0 1 0 0 0 0 1 0 4 1 4 2 9	0 0 0 1 1 0 0 0 1 1 0 4 0 7
Rib and vertebral configuration Cervical rib Cervical vertebrae 13th rib short Number of 14th ribs short Thoracolumbar vertebrae Pelvic girdle Delayed/Incomplete ossification/unossified Cranial Sternebrae Vertebrae cervical	short supernumerary full supernumerary additional short short supernumerary full supernumerary total 20 unilateral cranial shift unilateral caudal shift cranial centres 5th and/or 6th other total cervical thoracic sacrocaudal	2 0 0 2 26 0 26 1 0 1 3 33 10 37 0 0 1	1 0 0 0 16 1 17 0 1 1 5 13 3 13 0 2 0	1 1 1 1 29 1 29 1 0 3 3 7 9 13 0 2 0	2 0 0 0 31 0 31 0 0 0 9 12 8 15 1 4 0		1 0 0 2 13 0 13 1 0 1 2 17 7 18 0 0 1		1 0 0 0 9 1 10 0 1 1 3 6 2 6 0 2 0				1 1 1 1 13 1 13 1 0 3 2 5 5 8 0 1 0	2 0 0 0 14 0 14 0 0 0 6 8 7 10 1 4 0
Increased ossification Cranial Cervical vertebral centra	partially fused jugal to maxilla all ossified	1 19	0 14	2 18	2 10		1 9		0 6				2 10	2 7

Note: Individual fetuses/litters may occur in more than one category.

* Litter 28 excluded from group incidences – Suspected Day 21

Fetal examinations - minor visceral abnormality and necropsy findings - group incidences

Dose Group Dose (mg/kg/day)	Control 1 0	MDA 2 100	MDA 3 330	MDA 4 1000
		Fetuses					Litters
Group Number examined Total Number Affected		1 99 31	2* 87 30	3 95 29	4 101 14		1 21 18		2* 17 15				3 20 16	4 20 11
Visceral abnormalities Brain Eyes Thyroid Thymus Azygos vein Diaphragm Liver Kidney(s) Ureter(s) Testis(es) Umbilical artery Haemorrhages Head Abdomen General	dilated interventricular foramen variation in lens shape small lobe partially undescended lobe thymic remnant dilated thinning with liver protrusion fissured posterior caudate lobe small renal papilla dilated renal pelvis dilated/marked undescended malpositioned left brain abdominal cavity subcutaneous	0 1 0 9 0 1 1 0 0 0 0 0 2 18 4 0 0	3 0 1 1 0 0 0 0 1 0 1 0 0 16 4 5 2	0 1 0 9 2 0 0 1 1 1 1 1 2 9 6 1 2	0 1 0 2 0 0 1 0 0 0 0 0 0 8 2 2 0		0 1 0 6 0 1 1 0 0 0 0 0 2 13 4 0 0		2 0 1 1 0 0 0 0 1 0 1 0 0 10 4 5 2				0 1 0 8 2 0 0 1 1 1 1 1 2 9 6  1 1	0 1 0 2 0 0 1 0 0 0 0 0 0 7 2 2 0
Necropsy observations (external) Skin	shiny	0	1	0	0		0		1				0	0

Note: Individual fetuses/litters may occur in more than one category.

* Litter 28 excluded from group incidences – Suspected Day 21

Conclusions:

Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day produced no maternal toxicity or any effects on embryofetal survival or development.
Macroscopically, the kidneys of the dams given 330 or 1000 mg/kg/day were dark. This finding was considered to be related to the colour of the test item and therefore considered not to be adverse.
The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Executive summary:

The purpose of this study was to assess the influence of Bayscript Blaukomponente MDA on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Days 6-19 after mating) in the Han Wistar rat.

Three groups of 22 females received Bayscript Blaukomponente MDA at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, purified water, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, kidney weights and the gravid uterus weights were recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination, including cartilaginous examination.

Results

There was an increased incidence of dark kidneys in the treated groups, predominantly those given 330 or 1000 mg/kg/day, when compared with control in females killed on Day 20 of gestation.

Otherwise, there were no effects of treatment on maternal or litter parameters, including external fetal examination.

Conclusion

Administration of Bayscript Blaukomponente MDA to rats by oral gavage administration during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day produced no maternal toxicity or any effects on embryofetal survival or development.

Macroscopically, the kidneys of the dams given 330 or 1000 mg/kg/day were dark. This finding was considered to be related to the colour of the test item and therefore considered not to be adverse.

The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 414 study Bayscript Blaukomponente MDA was administered to rats by oral gavage during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day. Bayscript Blaukomponente MDA produced no maternal toxicity or any effects on embryofetal survival or development.

The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Toxicity to reproduction: other studies

Additional information

Bayscript Blaukomponente (konserviert) was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks. This GLP study was performed according to OECD guideline 407and GLP

Despite general toxic effects no pathological changes were observed at the reproductive organs in amle and female rats

Under the conditions described the no adverse observed effect level (NOAEL) for administration of BAYSCRIPT Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to mild to moderate effects on kidneys and minor changes in nerve roots.

With respect to reproductive organ toxitcity a NOAEL of 1000 mg/kgbw/day is established for male and female rats

Justification for classification or non-classification

In an OECD 414 study Bayscript Blaukomponente MDA was administered to rats by oral gavage during the organogenesis and fetal growth phase of pregnancy (Days 6-19 after mating) at 100, 330 or 1000 mg/kg/day. Bayscript Blaukomponente MDA produced no maternal toxicity or any effects on embryofetal survival or development.

The no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development was considered to be 1000 mg/kg/day. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.