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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase (ERBC): First allocation of animals to groups 13 October 2020 Start of experimental phase 02 December 2020 (first date of sample arrival at the Test Site) End of delegate phase (date PI signed the GLP statement) 31March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
474-870-9
EC Name:
-
Cas Number:
80156-97-4
Molecular formula:
Hill formula: C28H20ClN9Na4O16S5 CAS formula: C28H24ClN9O16S5.4Na
IUPAC Name:
tetrasodium 7-[(1E)-2-[2-(carbamoylamino)-4-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl]naphthalene-1,3,6-trisulfonate
Test material form:
solid: particulate/powder
Details on test material:
Batch No. LK2003715

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
The Sprague Dawley rat was the species and strain of choice because it is accepted by many
regulatory authorities and there are ample experience and background data on this species
and strain.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
softened by reverse osmosis
Details on exposure:
The test item was administered orally, by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
The dose levels of 100, 300 and 1000mg/kg bw/day were selected by the Sponsor based on information from previous studies.
Analytical verification of doses or concentrations:
yes
Remarks:
Samples of the preparations prepared during the first and last week of treatment of the current study were analysed to check the concentration. Final results for all levels were within the acceptability limits for concentration (90-110%).
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC Study no. A4042). The stability of the preparations at 5 to 100mg/mL was assessed at:
– for 28 hours at room temperature;
– for 10 days at +2-8°C.

Samples of the preparations prepared during the first and last week of treatment of the current study were analysed to check the concentration.
Chemical analysis was carried out by the Analytical Chemistry Department. Results of preparation analyses are presented in Addendum 3 of this report. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (90-110%).
The validated software used for this activity was SkanIt version 6.0.0.44.
Details on mating procedure:
The females were paired with male rats. Females were paired 1 to 1 in the home cage of the male and left overnight. Allocation was performed to prevent any stock male from providing more than one mated female in each treated group, except on two occasions.
Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records will be maintained.
Duration of treatment / exposure:
Daily from Day 3 to Day 19 post coitum
Frequency of treatment:
daily
Duration of test:
20 days, with 17 days of treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control group
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
Each group comprised 25 mated female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
Animal management
Animal supply and acclimatisation
A total of 130 Sprague Dawley virgin female rats, 9 weeks old (209 to 226 g) were received from Charles River Italia S.p.A., Calco (Lecco), Italy. The male rats used were from the same supplier, and were at least 11 weeks old (at least 340 g). Mating of siblings was avoided. After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of 23 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal husbandry
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. Before and after the pairing period, the animals were housed no more than 5 of one sex in polysulfone solid bottomed cages, measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the pairing period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) with certified dietary levels of phytooestrogen (genistein level of 124mg/kg) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 to a cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Administration of test item
The test item was administered orally by gavage at a dose volume of 10mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Duration of treatment
All animals were dosed once a day from Day 3 through Day 19 post coitum. Caesarean section was done on Day 20 post coitum.

Examinations

Maternal examinations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. A complete necropsy was performed in all cases.

Clinical signs
All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded from allocation until sacrifice.

Observations of the cage tray
Observations of the cage tray was performed daily starting from Day 8 post coitum until termination.

Body weight
All animals were weighed on Days 0, 3, 5, 9, 12, 15, 18 and 20 post coitum.

Food consumption
Food consumption was measured on Days 3, 5, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

Clinical pathology investigations - Dams
Blood collection - Thyroid hormone determination (T3, T4 and TSH)
Blood collection by random selection (order of collection equalised between groups) was performed, for hormone determination, from all females at termination, on Day 20 post coitum. Blood samples (approximately 0.8 mL) for hormone determination were withdrawn from the sublingual vein under light isoflurane anaesthesia.

Terminal studies
Euthanasia
The animals were killed on Day 20 post coitum and necropsied as detailed below. Animals
were euthanised with carbon dioxide and subjected to necropsy, supervised by a pathologist.
All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by
hypothermia.

Necropsy - Dams
The clinical history of the animal was studied and a detailed post mortem examination was
conducted (including examination of the external surface and orifices). The evaluation of the dams during necropsy procedures was performed applying the randomization across treatment groups.

Organ weight - Dams
From all females completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to brain weight was calculated for each animal.
Ovaries and uterine content:
The ovaries were examined to determine:
– number of corpora lutea

The uteri and their content were examined to determine:
– Gravid uterine weight, including the cervix;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number of intra-uterine deaths;
– gross evaluation of placentae;
– internal foetal sex determination in each foetuses allocated to the skeletal examination;
– anogenital distance (AGD) in all live foetuses

Intra-uterine deaths were classified as:
– Early resorptions: only placental remnants visible.
– Late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.

Fetal examinations:
Examination of foetuses
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination.

Skeletal and fixed-visceral examinations were performed in all groups.

Structural deviation were classified as follows:
Malformations: Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies: Minor abnormalities that are detected relatively frequently.
Variants: A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of theWilliams test. The criterion for statistical significance was
p<0.05. The mean values, standard deviations and statistical analysis were calculated from
the actual values in the computer without rounding off.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss% = no. of corpora lutea−no. of implantations/no. of corpora lutea×100

Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss% = no. of implantations−no. of live foetuses/no. of implantations×100

Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss% = no. of corpora lutea−no. of live foetuses/no. of corpora lutea×100

Sex ratios of the foetuses were calculated as the percentage of males per litter.
Anogenital distance (AGD): The AGD of each live foetuses was measured on Day 20 post
coitum. The AGD was normalized to the cube root of body weight collected on Day 20 post
coitum.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were recorded.
Mortality:
no mortality observed
Description (incidence):
No mortality of females occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in bodyweight/bodyweight gain were seen between groups throughout
the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was comparable between groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted in thyroids as well as all abnormalities.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No changes were observed in brain and thyroid weight of treated animals, when compared
to the controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment-related macroscopic observation, consisted of yellow colour of the placenta,
observed in most of high dose females. This finding was considered due to the colour of
the test item.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone determination
Anogenital distance

Details on results:
Thyroid hormone determination: The serum levels of T3, T4 and TSH did not differ between treated and control groups.
Anogenital distance: Anogenital distance (AGD): The AGD of each live foetuses was measured on Day 20 post
coitum. The AGD was normalized to the cube root of body weight collected on Day 20 post
coitum.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were sacrificed at termination and were found pregnant with the exception of
two females (one at 100 mg/kg bw/day and one at 300 mg/kg bw/day).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Compared to the control, no alterations were seen in the anogenital distance performed in
foetuses maternally exposed to the test item.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were seen at external examination of foetuses.
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment with the test item Reactive Yellow 176 did not cause maternal toxicity and did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg bw/day.
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level)
for maternal and developmental toxicity is considered to be 1000 mg/kg bw/day or above.
Executive summary:

The effects of Reactive Yellow 176 were investigated after oral administration in female rats during pregnancy and on embryo-foetal development.
Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each.
All females were administered by oral gavage during the gestation period from Day 3 through Day 19 post coitum at 100, 300 and 1000 mg/kg bw/day and 10 mL/kg body weight as the dose volume. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation. Control females received the vehicle (water, softened by reverse osmosis) at the same dose volume during the same treatment period.
Mortality check, clinical signs, body weight and food consumption were recorded during the in vivo phase. Before despatch to necropsy (Day 20 post coitum), blood samples were collected for hormones determination of T3, T4 and TSH. At term, females were caesarean sectioned on Day 20 post coitum and subjected to detailed post mortem examination.
The thyroid and brain were weighed and preserved in fixative. Thyroid was examined histopathologically. Gravid uterus was weighed and the uterine content examined for the number of implantations sites, intrauterine deaths and live foetuses. The ovaries were also examined and the corpora lutea counted. Live foetuses were weighed, sexed and observed for external abnormalities. After killing the foetuses were then processed for skeletal and fixed visceral examinations.


No mortality of females occurred during the study.
All females were sacrificed at termination and were found pregnant with the exception of two females (one at 100 mg/kg bw/day and one at 300 mg/kg bw/day). The final number of pregnant females on Day 20 post coitum was 25 in the control group, 24 in low- (100 mg/kg bw/day) and mid-dose levels (300 mg/kg bw/day) and 25 in the high dose group (1000 mg/kg bw/day).



No treatment-related clinical signs were recorded. No significant changes in bodyweight or bodyweight gain were seen between groups throughout the study. Food consumption was comparable between groups.


The serum levels of T3, T4 and TSH did not differ between treated and control groups.
Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment. Caesarian section data, litter data and sex ratios were unaffected by treatment. Compared to the control, no alterations were seen in the anogenital distance performed in foetuses maternally exposed to the test item.


No changes were observed in brain and thyroid weight of treated animals, when compared to the controls. Treatment-related macroscopic observation, consisted of yellow colour of the placenta, observed in most of high dose females. This finding was considered due to the colour of the test item.
No treatment-related changes were noted in thyroids as well as all abnormalities during histophathological examination.
No treatment-related findings were recorded at external, visceral or skeletal examination of foetuses.


In conclusion, treatment with the test item Reactive Yellow 176 did not cause maternal toxicity and did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg bw/day.
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity is considered to be 1000 mg/kg bw/day or above.