Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Principles of method if other than guideline:
This study was designed to assess the influence of Macrolex Rot EG, a colorant for plastics, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley rat.
One group of 20 females received Macrolex Rot EG at a dose of 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, polyethylene glycol 400 (PEG 400) at the same volume dose as the treated group. 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 and the gravid uterine weight recorded and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test item: Macrolex Rot EG.
Test item identity Macrolex Red EG.
(including alternative names): 8,9,10,11-tetrachloro-12H-phthaloperin-12-one.
CAS number: 20749-68-2.
Empirical formula: C18H6Cl4N2O.
Molecular mass: 408.1 g/moL.
Appearance: Red powder.
Storage conditions: At ambient temperature.
Supplier: Sponsor.
Purity: 99.6%.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) rat
Details on test animals and environmental conditions:
Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals ordered 46 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization 40 days from arrival on site before commencement of pairing.
Age of the animals at the start of the
study (Day 0 of gestation) 69 to 75 days
Weight range of the animals at the
start of the study (Day 0 of gestation) 245 to 309 g

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock 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 temporary tail mark until the day of positive evidence of mating and then 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 minimize entry of external biological and chemical agents.
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%. There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
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, which was changed at appropriate intervals each week.
Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

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

Diet Supply
Diet SDS VRF1 Certified pelleted diet (from the time the animals are allocated to the study).
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 at appropriate intervals.
Availability Non-restricted.

Dose Administration
Study Design and Identity of Treatment Groups
Mating Day of study: 0
Treatment Day of study: 6 to 19
Necropsy Day of study: 20

The study consisted of one control and one treated group identified as follows:
Group Treatment Dose (mg/kg/day) Number of animals Animal numbers
1 Control 0 20 1-20
2 Macrolex Rot EG 1000 20 21-40


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Mating Day of study: 0
Treatment Day of study: 6 to 19
Necropsy Day of study: 20
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity Homogeneity and stability of the test material in the vehicle was determined in GLP compliant Sponsor study.
In that study the stability over the range 20 to 200 mg/mL was confirmed as eight days at ambient temperature.
Achieved concentration Samples of each formulation prepared for administration in Weeks 1 and the last week of treatment were analyzed for achieved concentration of the test item.
Details on mating procedure:
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
From day 6 to 19 (14 days)
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Macrolex Rot EG
No. of animals per sex per dose:
The study consisted of one control and one treated group identified as follows:
Group Treatment Dose (mg/kg/day) Number of animals Animal numbers
1 Control 0 20 1-20
2 Macrolex Rot EG 1000 20 21-40
Control animals:
yes, concurrent vehicle
Details on study design:
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 acclimatization 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:
Pre-dose observation.
One to two 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 0, 5, 12, 18 and 20 after mating to monitor general health.

Also due to the red powdery appearance of the test item it was considered necessary to perform bedding observations as there was a potential for the faeces to be of abnormal color. Each animal was recorded on Day 10, 14, 18 and 20 after mating to monitor any changes to the faeces.

Body Weight
The weight of each adult was recorded on Days 0, 3, 6 and daily thereafter.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Carbon dioxide asphyxiation.
Method of kill for fetuses Chilling on a cool plate (approximately 0°C).

Necropsy
All adult animals 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.
Schedule Animals were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.

Reproductive Assessment
The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

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 double 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 double stained fetuses Assessed for skeletal and cartilage development and abnormalities.

Data Evaluation
This report only contains serial observations pertaining to the gestation period (from Day 0 after mating).

Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data. Summary tabulated data was normally restricted to data derived from females/litters with live young at Day 20 after mating. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.

Throughout the report the following abbreviations are used:
F Female
M Male
N Number contributing to the mean (normally the number of animals/litters)
SD Standard deviation

The death code in the appendices has the following meaning:
T Scheduled kill; pregnancy confirmed

Standard deviations have not been calculated for derived data, such as levels of pre- and post implantation loss, or for the incidence of resorbing fetuses where the distribution of these findings commonly does not conform to the normal statistical distribution.

Serial Observations
Clinical Observations
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.
There were no signs associated with dosing observed and therefore these data are not presented.

Body Weight
Group mean weight changes were calculated from the weight changes of individual animals. Weight changes were calculated and body weights were plotted graphically with respect to Day 6 of gestation.
Adjusted body weights on Day 20 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-20 were also presented, after being adjusted for the contribution of the gravid uterus.

Food Consumption
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values.

Terminal Investigations
Reproductive Assessment
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.

Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.
Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.
Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

In the Fetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.

Statistical Analysis
The following data types were analyzed at each time-point separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparison was performed:
Group 1 vs 2

A sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data.


Examinations

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 acclimatization 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:
Pre-dose observation.
One to two 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 0, 5, 12, 18 and 20 after mating to monitor general health.

Also due to the red powdery appearance of the test item it was considered necessary to perform bedding observations as there was a potential for the faeces to be of abnormal color. Each animal was recorded on Day 10, 14, 18 and 20 after mating to monitor any changes to the faeces.

Body Weight
The weight of each adult was recorded on Days 0, 3, 6 and daily thereafter.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Necropsy
All adult animals 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.
Schedule Animals were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.
Ovaries and uterine content:
Reproductive Assessment
The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Fetal examinations:
Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.
Group mean values and SD were calculated using individual litter mean values.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.
Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

In the Fetal examinations appendix, observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.
Statistics:
Statistical Analysis
The following data types were analyzed at each time-point separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight

The following comparison was performed:
Group 1 vs 2

Av: Pretreatment comparison of all groups using Analysis of variance followed by pairwise t-tests
Tt: Treated groups compared to Control using the t-test.
Wa: Treated groups compared with Control using Wald’s test
Wc: Treated groups compared to Control using the Wilcoxon rank sum test.
* p<0.05
** p<0.01
Indices:
Terminal Investigations
Reproductive Assessment
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:
Historical control data for skeletal/visceral abnormalities on Crl:CD(SD) rats - the rat strain used in the study - are available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Treatment of Macrolex Rot EG to pregnant female Sprague Dawley rats from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.
Mortality:
no mortality observed
Description (incidence):
Treatment of Macrolex Rot EG to pregnant female Sprague Dawley rats from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain during gestation were considered to be unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption during gestation were considered to be unaffected by treatment.
Food efficiency:
not specified
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):
Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) during gestation - were unaffected.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At macroscopic examination, red coloration in the stomach, rectum, ileum, colon, caecum or on the tail or pinnae was observed in a small number of females (5/20) treated at 1000 mg/kg/day. There were no other maternal findings observed at macroscopic examination that were considered to be related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Based on the results of this study, it is concluded that in the absence of any evidence for general systemic toxicity, that the no observed adverse effect level was 1000 mg/kg/day.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Litter size and pre-/post-implantation loss were unaffected by treatment.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter size and pre-/post-implantation loss were unaffected by treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Embryo-fetal survival was unaffected by treatment at 1000 mg/kg/day with mean numbers of implantations, resorptions, live young, percentage sex ratio and pre-/post-implantation loss being similar to controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Embryo-fetal survival was unaffected by treatment at 1000 mg/kg/day with mean numbers of implantations, resorptions, live young, percentage sex ratio and pre-/post-implantation loss being similar to controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
Embryo-fetal survival was unaffected by treatment at 1000 mg/kg/day with mean numbers of implantations, resorptions, live young, percentage sex ratio and pre-/post-implantation loss being similar to controls.
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):
Embryo-fetal survival was unaffected by treatment at 1000 mg/kg/day with mean numbers of implantations, resorptions, live young, percentage sex ratio and pre-/post-implantation loss being similar to controls.
Other effects:
no effects observed
Details on maternal toxic effects:
No effects observed.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Absence of adverse toxic effects at the highest applied dose of 1000 mg/kg day

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Group mean placental, litter and fetal weights (male, female and overall) were unaffected by treatment at 1000 mg/kg/day.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Group mean placental, litter and fetal weights (male, female and overall) were unaffected by treatment at 1000 mg/kg/day. Litter size was unaffected by treatment at 1000 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Litter size was unaffected by treatment at 1000 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Embryo-fetal survival was unaffected by treatment at 1000 mg/kg/day with mean numbers of implantations, resorptions, live young, percentage sex ratio and pre-/post-implantation loss being similar to controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Group mean placental, litter and fetal weights (male, female and overall) were unaffected by treatment at 1000 mg/kg/day. Litter size was unaffected by treatment at 1000 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
At macroscopic examination, red coloration in the stomach, rectum, ileum, colon, caecum or on the tail or pinnae was observed in a small number of females (5/20) treated at 1000 mg/kg/day. There were no other maternal findings observed at macroscopic examination that were considered to be related to treatment.
The red content / staining observed in-life and at macroscopic examination, in females treated at 1000 mg/kg/day was considered discolouration caused by the dye itself and was not considered to represent toxicity.
Skeletal malformations:
no effects observed
Description (incidence and severity):
At 1000 mg/kg/day there was a slightly increased incidence of the minor abnormality, short supernumerary 14th ribs compared to concurrent control and historical control data ranges. In isolation, this finding was considered not to represent an adverse effect of treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
At 1000 mg/kg/day there was a slightly increased incidence of the minor abnormality malpositioned testis(es) (defined as one or both testes lying over the midline of the bladder at the time of examination) compared to concurrent control and historical control data ranges. This minor finding was considered not to represent an adverse effect of treatment on fetal development since:
- Two testes had developed as expected in all male fetuses.
- The evaluation of the position of the testes represents an evaluation at a snapshot in time of a mobile, non-tethered organ which at any given point in time could be at any position in the abdomen. Between Days 19 and 21 of gestation the testes are normally on either side of the bladder, therefore any deviation from this is recorded but is considered as a biological variation in position. After birth, the testes remain mobile and non tethered in rats: the testes normally lose their position in the abdomen and descend into the scrotum at approximately 15 days following birth. Testes that have not or do not descend may be retained in the abdominal cavity or the inguinal canal.

It is important to note that throughout life male rats normally have open inguinal canals with a functional cremaster muscle that allows the testes (testicles) to migrate easily in and out of the abdominal cavity for both protection and temperature control.
For clarification, in addition to the fetuses that had malpositioned testis(es) one fetus in the control group had the finding ‘undescended testes’. This is defined as the testes positioned slightly higher in the abdomen with a clear gap evident between the bottom of the testes and the top of the bladder.
Other effects:
no effects observed
Description (incidence and severity):
Based on the results of this study, it is concluded that in the absence of any evidence for general systemic toxicity or adverse effects on embryo-fetal survival, growth and development, that the no observed adverse effect level (NOAEL) was 1000 mg/kg/day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of adverse toxic effects at the highest applied dose of 1000 mg/kg day

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Body weight - group mean values (g) during gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

 Group/

sex

 Day

0

  Day

3

 Day

6

  Day

7

  Day

8

Day

9

 Day

10

Day

11

Day

12

  Day

13

 Day

14

 Day

15

 Statistical test:

 1 F

 Mean

SD

N

 Av

269

16.4

20

Av

287

14.7

20 

Av

300

15.6

20 

Tt

302

13.9

20 

Tt

307

15.4

20 

Tt

313

15.9

20 

Tt

319

14.7

20 

Tt

323

15.3

20 

Tt

328

14.8

20 

Tt

331

15.9

20 

Tt

336

15.9

20 

Tt

345

15.8

20 

 2 F

  Mean

SD

N

268

12.8

20

287

13.5

20 

299

15.4

20

301

14.4

20

306

15.4

20

312

18.6

20

318

17.7

20 

323

16.8

20 

331

17.3

20

334

17.5

20

340

18.3

20

349

19.5

20 

Body weight - group mean values (g) during gestation (continued)

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

  

Group/

sex

 

 Day

16

 Day

17

 Day

18

Day

19 

 Day

20

 Change

0 -6

 Change

6 -19

Change

0 -20 

Statistical test:

 1 F

  

Mean

SD

N

Tt

354

16.2

20 

Tt

367

21.7

20 

Tt

384

23.5

20 

Tt

397

24.5

20 

Tt

414

28.0

20 

Av

31

6.0

20 

Tt

97

18.4

20 

Tt

145

24.1

20 

 2 F

Mean

SD

N

360

19.5

20 

 373

20.7

20

390

21.6

20 

406

22.1

20 

423

22.6

20 

 31

8.5

20

107*

12.5

20 

155

18.2

20 

Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g) during gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

  

Group/

sex

 

 Body weight

on Day 6

 Terminal

Body weight

on Day 20

Body weight

Change Days 6 -20 

 Gravid

Uterine

Weight

Adjusted

Body weight

Day 20 

Adjusted

Body weight change

Days 6 -20 

Statistical test:

 1 F

  

Mean

SD

N

 Av

300

15.6

20

Tt

414

28.1

20 

Tt

114

22.6

20 

Tt

87

23.4

20 

 Tt

327

17.0

20

Tt

27

12.8

20 

 2 F

 Mean

SD

N

299

15.4

20 

424

22.7

20 

 125

13.7

20

 92

13.0

20

331

21.4

20 

33

10.1

20 

Food consumption - group mean values (g/animal/day) during gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

  

Group/

sex

 

Day

0 -2 

 Day

3 -5

Day

6 -9 

Day

10 -13 

Day

14 -17 

Day

18 -19 

Statistical test:

 1 F

  

Mean

SD

N

Av

22

1.6

20 

Av

24

2.0

20 

Tt

24

2.2

20 

Tt

25

2.8

20 

Tt

27

2.8

20 

Tt

28

3.5

20 

 2 F

Mean

SD

N

23

2.2

20 

24

2.1

20 

24

2.5

20 

 26

2.0

20

28

2.6

20 

28

3.9

20 

Macropathology - group distribution of findings on Day 20 of gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

 Tissue and Finding

 Group/Sex:

Number Examined:

 1F

20

 2F

20

General comments

Fur stained

   1  1

Skin

Hair loss

   2  2

Liver

Misshappen

Small

 

1

1

0

Caecum

Contents tinted red

 

 0

 2

Colon

Contents tinted red

 

 0

 2

Ileum

Contents tinted red

 

 0

 1

Rectum

Contents tinted red

 

 0

 3

Stomach

Contents tinted red

 

 0

 1

Tail

stained

 

 0

 1

Placenta

Pale

Small

 

0

0

1

Litter data - group mean values on Day 20 of gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

Group/

Sex

 

Corpora

Lutea 

 Implantations

Resorptions

Early Late Total       

 Male

 Live Young

Female

 Total

 Sex ratio

(%M)

    Implantation Loss (%)

Pre- Post-

  

Statistical test:

 1 F

Mean

SD

N

Tt

17.2

2.35

20 

Wc

15.8

3.65

20 

Wc

0.9

20 

Wc

0.1

20

Wc

1.0

20

 Tt

7.6

3.05

20

Tt

7.3

3.29

20

Tt

14.8

4.21

20 

Wa

50.6

20 

Wa

8.6

20 

Wa

7.8

20 

 2 F

Mean

SD

N

18.0

1.59

20 

16.6

1.19

20 

0.9

20

0.1

20

1.0

20 

7.6

2.23

20 

8.0

2.36

20 

15.6

2.30

20 

 48.7

20

7.9

20 

5.8

20 

Placental, litter and fetal weights - group mean values (g) on Day 20 of gestation

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

 Group/

Sex

 

 Placental

Weight

 Litter

Weight

 Litter

Size

 Male Fetal

Weight

 Female Fetal

Weight

 Overall Fetal

Weight

  

Statistical test:

 1 F

 

Mean

SD

N

Tt

0.53

0.089

20 

Tt

53.18

15.373

20 

 Tt

14.80

4.213

20

 Wc

3.68

0.468

20

Tt

3.52

0.344

20 

Wc

3.60

0.387

20 

 2 F

Mean

SD

N

0.53

0.050

20 

 56.43

9.201

20

15.60

2.303

20 

3.72

0.199

20 

3.52

0.191

20 

3.61

0.187

20 

Fetal examinations - major abnormality findings - group incidences

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

Group

Number examined

Total Number Affected

 

Fetuses

1

296

Fetuses

2

312

Litters

1

20

Litters

2

20

Head

Visceral

External

Medially displaced cochlea

Domed head

1

1

0

0

1

1

0

0

Cervical/Thoracic

Skeletal

Partially spilt sternum

 

1

 

0

 

1

0

Appendicular

Skeletal

Bent long bone(s)

 

1

 

0

 

1

 

0

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

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

Group

Number Examined

 Fetuses

1

149

Fetuses

2

156 

 Litters

1

20

Litters

2

20 

Minor skeletal abnormalities

Cranial interparietal fissure(s)

Cartilaginous centrum unfused

Ribs medially thickened/kinked

Sternebrae partially fused

Sternebrae misshapen

Costal cartilage hole in xiphoid

Costal branched xiphoid

Appendicular misshapen cranial margin scapula(e)

Total affected by one or more of the above

1

1

2

1

1

0

1

1

0

0

1

0

0

2

1

1

1

1

2

1

1

0

1

1

0

0

1

0

0

2

1

1

Rib and vertebral configuration

Cervical rib short supernumerary

13th rib short

Number of 14th ribs short supernumerary

Number of 14th ribs total

Thoracolumbar vertebrae

Pelvic girdle unilateral caudal shift

1

0

5

5

1

2

1

14

14

0

1

0

5

5

1

2

1

8

8

0

Delayed/Incomplete ossification/unossified

Cranial cranial centres  

Cranial presphenoid  

Cranial hyoid

Sternebrae 5th and/or 6th

Sternebrae other

Sternebrae total

Vertebrae cervical

Vertebrae thoracic

Vertebrae lumbar

Vertebrae sacrocaudal

Vertebrae caudal

Ribs any

Appendicular pelvic bones

Appendicular metatarsals

20

2

8

82

8

82

8

16

1

11

0

1

14

14

1

17

89

6

89

3

4

1

12

1

0

11

10

1

6

17

6

17

6

11

1

6

0

1

6

8

1

9

19

4

19

3

3

1

6

1

0

6

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

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

Group :                    1                     2

Compound :       Control       Macrolex Rot EG

Dose (mg/kg/day) :  0                      1000

Group

Number Examined

Total Number affected

Fetuses

1

147

17

Fetuses

2

156

17 

Litters

1

20

11 

Litters

2

20

12 

Visceral abnormalities

Brain dilated ventricles

Brain misshapen pituitary

Thyroid small lobe

Thymus partially undescended lobe

Thymus thymic remnant

Liver malpositioned cleft

Kidney(s) small renal papilla

Ureter(s) dilated

Testis(es) undescended

Testis(es) malpositioned

Umbilical artery left 1 0 1 0

Haemorrhages

Head brain

Neck/Thorax thoracic cavity

Abdomen abdominal cavity

Abdomes liver lobes

1

1

1

5

1

1

0

0

1

3

1

2

0

1

0

0

0

2

1

0

1

1

0

6

0

4

1

3

1

1

1

4

1

1

0

0

1

3

1

2

0

1

0

0

0

2

1

0

1

1

0

6

0

2

1

3

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

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it is concluded that in the absence of any evidence for general systemic toxicity or adverse effects on embryo-fetal survival, growth and development, that the no observed adverse effect level was 1000 mg/kg/day (limit dose, highest dose tested).
Executive summary:

Method: One group of 20 females received Macrolex Rot EG at a dose of 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, polyethylene glycol 400 (PEG 400) at the same volume dose as the treated group. 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 and the gravid uterine weight recorded and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results: Treatment of Macrolex Rot EG to pregnant female Sprague Dawley rats from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg/day was well tolerated and elicited no deaths and no toxicity-related changes in clinical condition of the adult females.

Body weight gain and food consumption during gestation were considered to be unaffected by treatment.

A small incidence of females treated at 1000 mg/kg/day (5/20) were observed to have red staining of the tail; in addition red faeces were observed in all cages of females in this group from Day 10 of gestation.

At macroscopic examination, red coloration in the stomach, rectum, ileum, colon, caecum or on the tail or pinnae was observed in a small number of females (5/20) treated at 1000 mg/kg/day.

There were no other maternal findings observed at macroscopic examination that were considered to be related to treatment.

The red content / staining observed in-life and at macroscopic examination, in females treated at 1000 mg/kg/day was considered discolouration caused by the dye itself and was not considered to represent toxicity.

Litter size and pre-/post-implantation loss were unaffected by treatment.

Group mean placental, litter and fetal weights were unaffected by treatment at 1000 mg/kg/day.

At 1000 mg/kg/day there was a slightly increased incidence of the minor fetal abnormalities, short supernumerary 14th ribs and malpositioned testis(es) compared to concurrent control and the historical control data ranges. These abnormalities however are unrelated, were seen in isolation and consequently were considered not to be an adverse response to treatment.

Conclusion: Based on the results of this study, it is concluded that in the absence of any evidence for general systemic toxicity or adverse effects on embryo-fetal survival, growth and development, that the no observed adverse effect level was 1000 mg/kg/day (highest dose tested).