Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

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:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jun 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
EC Number:
271-176-6
EC Name:
Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate
Cas Number:
68516-73-4
Molecular formula:
C34H32N6O12
IUPAC Name:
tetramethyl 2,2'-{1,4-phenylenebis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}diterephthalate
Test material form:
solid: nanoform, no surface treatment
Details on test material:
- State of aggregation:
- Particle size distribution:

- Shape of particles:
- Surface area of particles: BET = 46.3 m2/g
- Crystal structure: crystalline
- Coating: none
- Batch: 13007010
- solid yellow
Specific details on test material used for the study:
Content: 95.7%
Storage stability: Expiry date: 08 Nov 2028
Physical state / appearance: solid / yellow
Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: ca 190g
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination 6 am to 6 pm)

IN-LIFE DATES: From: 19/20/21 Oct (Cohort 1/2/3, resp.) 2021 To: 8/9/10 Nov (Cohort 1/2/3, resp.) 2021

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Tween 80
Remarks:
0.5% in deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Beginning of administration and thereafter at regular intervals
- Volume applied: 10 ml/kg bw/day
- Concentration: 1.00, 3.00, 10.00 g/100 ml for test groups 100, 300, 1000 mg/kg bw/day, respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate
study in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in drinking water at room temperature over a period of 7
days had been verified prior to the start of the study in a similar batch.
Samples of the test substance preparations were sent thrice to the analytical laboratory for
verification of the concentrations. Due to difficulties in the manufacture of the test substance preparations, the attained results of homogeneity and
concentration control analyses of samples taken during the in-life phase were inconclusive. Therefore, a third set
of homogeneity/concentration control samples were generated after the end of the in-life phase, using the same
procedures and under the same conditions as the previous samples.
The samples were also used to verify the homogeneity of the low- and high-concentrations (100 and 1000 mg/kg bw/d). For the latter purpose three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C).
Details on mating procedure:
Time-mated
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
daily
Duration of test:
from implantation to one day prior to the expected day of parturition
Doses / concentrationsopen allclose all
Dose / conc.:
78 mg/kg bw/day (actual dose received)
Remarks:
nominal 100 mg/kg bw
Dose / conc.:
208 mg/kg bw/day (actual dose received)
Remarks:
nominal 300 mg/kg bw
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
nominal 1000 mg/kg bw
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Result of OECD 422 study
- Rationale for route: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Due to technical reasons, the study was carried out in 3 cohorts.
- Reason for species selection: The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked: morbidity, pertinent behavioral changes and/or signs of overt toxicity

CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
- Clinical observations checked: mortality

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD20
- Blood samples obtained: Yes, from all animals
- Anesthesia: Yes (Isoflurane)
- Organs examined: Thyroid glands (with parathyroid glands), All paired organs were weighed together (left and right).
- Histopathology: Yes, Thyroid glands

THYROID HORMONES
- Parameters checked: Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes
Blood sampling:
On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Anogenital distance: Yes
- Fetuses were sexed, weighed, condition of placenta checked and umbilical cords, fetal membranes, and fluids were examined.
Statistics:
DUNNETT-test, FISHER'S EXACT test, WILCOXON-test, KRUSKAL-WALLIS test.
* for p < 0.05
** for p < 0.01
Indices:
The conception rate, The preimplantation loss, The postimplantation loss, anogenital index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Yellowish discolored feces were recorded for each eight mid- and high-dose females (300 and 1000 mg/kg bw/d) from GD 8, furthermore, for all mid- and high-dose females from GD 10
onwards. Whereas up to 15 (out of 25) low-dose females (100 mg/kg bw/d) where affected from GD 12 onwards until terminal sacrifice (GD 20). This feces discoloration mirrors the presence of the yellowish test substance in the gastrointestinal tract. An adverse, toxic effect is not assumed.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. No weight changes were noted in in the thyroid glands.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A yellowish discolored content of the duodenum was recorded in 9 (out of 25) high-dose females, while a discolored content of the rectum as well as of the caecum was seen in 3 highdose females, respectively. These discolorations mirror the presence of the test substance in the gastrointestinal tract. They are considered as non-adverse, causing no toxic effects by themselves.
Additionally, two spontaneous findings were noted in individual females of test groups 2 and 3 (300 and 1000 mg/kg bw/d). A diaphragmatic hernia was seen in mid-dose female No. 61, while a dilated renal pelvis was recorded for mid-dose female No. 66 and high-dose female No. 94.
Since these findings occurred in single females without a dose response relationship, they were assessed as incidental and not treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

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:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
maternal developmental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

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
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
- The mean placental weights of the low-, mid- and high-dose groups were comparable to the corresponding control group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
prenatal development
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Total external malformations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
295		 25
287		 25
292		 25
300
Fetal incidence N (%) 0.0 1 (0.3) 0.0 0.0
Litter incidence N (%) 0.0 1 (4.0) 0.0 0.0
Affected fetuses/litter Mean% 0.0 0.3 0.0 0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One female fetus in test group 1 showed micromelia.

Table 2: Total external variations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
295		 25
287		 25
292		 25
300
Fetal incidence N (%) 0.0 1 (0.3) 0.0 0.0
Litter incidence N (%) 0.0 1 (4.0) 0.0 0.0
Affected
fetuses/litter		 Mean% 0.0 0.3 0.0 0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One external variation in one single fetus (No. 46-09) was recorded in test group 1 (100 mg/kg bw/d), i.e. limb hyperextension. The incidence of this single finding was not statistically significantly different from control and there is no dose response relationship visible. This finding can also be found in the historical control data at comparable incidences.

Table 3: Total external unclassified observations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
295		 25
287		 25
292		 25
300
Fetal incidence N (%) 0.0 0.0 2 (0.7) 1 (0.3)
Litter incidence N (%) 0.0 0.0 2 (8.0) 1 (4.0)
Affected
fetuses/litter		 Mean% 0.0 0.0 0.6 0.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

One external unclassified observation (placentae fused) was recorded in two mid-dose and one high-dose fetuses without relation to dose. This is a common finding which can be found in the historical control data, thus, it is considered as not treatment-related.

Table 4: Total soft tissue variations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
140		 25
137		 25
140		 25
142
Fetal incidence N (%) 4 (2.9) 5 (3.6) 4 (2.9) 4 (2.8)
Litter incidence N (%) 4 (16) 5 (20) 4 (16) 4 (16)
Affected
fetuses/litter		 Mean% 2.4 3.3 2.9 2.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Three soft tissue variations were detected, i.e. short innominate in one control fetus, dilated renal pelvis and dilated ureter in several fetuses of all test groups including the control. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences.

Table 5: Individual fetal skeletal malformations

Test group Dam No.-Fetus No., Sex Finding
0 (0 mg/kg bw/d) none  
1 (100 mg/kg bw/d) 30-12 Fa)
50-09 F		 multiple skeletal malformations
cleft sternum
2 (300 mg/kg bw/d) none  
3 (1000 mg/kg bw/d) 81-01 M shortened scapula

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a) fetus with additional external malformation

Table 6: Total skeletal malformations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
155		 25
150		 25
152		 25
158
Fetal incidence N (%) 0.0 2 (1.3) 0.0 1 (0.6)
Litter incidence N (%) 0.0 2 (8.0) 0.0 1 (4.0)
Affected fetuses/litter Mean% 0.0 1.2 0.0 0.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7: Total fetal skeletal variations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
155		 25
150		 25
152		 25
158
Fetal incidence N (%) 152 (98) 143 (95) 151 (99) 151 (96)
Litter incidence N (%) 25 (100) 25 (100) 25 (100) 25 (100)
Affected
fetuses/litter		 Mean% 98.2 95.4 99.2 95.2

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

Table 8: Total unclassified cartilage observations

    Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d
Litter Fetuses N N 25
155		 25
150		 25
152		 25
158
Fetal incidence N (%) 113 (73) 117 (78) 104 (68) 130 (82)
Litter incidence N (%) 25 (100) 24 (96) 24 (96) 25 (100)
Affected
fetuses/litter		 Mean% 72.6 77.1 70.1 81.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Table 9:Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding Test group 0
0 mg/kg bw/d		 Test group 1
100 mg/kg bw/d		 Test group 2
300 mg/kg bw/d		 Test group 3
1000 mg/kg bw/d		 HCD
Mean % (range)
Incomplete ossification of thoracic centrum; unchanged cartilage 0.0 0.7 0.0 2.0* 0.4
(0.0 - 1.6)
Incomplete ossification of sacral arch;
cartilage present		 0.0 1.8* 2.0 0.8 0.9
(0.0 - 3.8)
Misshapen sternebra; unchanged cartilage 19.5 28.4 16.0 29.2* 24.3
(11.4 - 32.4)
Supernumerary rib (14th); cartilage not present 48.6 63.0** 56.6 51.5 51.0
(37.5 - 64.9)
Incomplete ossification of tuberositas deltoidea; cartilage present 0.0 0.7 0.8 2.0* 0.3
(0.0 - 1.8)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

** = p <= 0.01 (Wilcoxon-test [one-sided])

The findings ‘incomplete ossification of sacral arch (cartilage present)’, ‘misshapen sternebra (unchanged cartilage)’ and ‘supernumerary rib (14th) (cartilage not present)’ were not related to dose and the mean values were clearly inside the historical control ranges. Therefore, these findings are assessed as not treatment-related. There was an increase in incidence at 1000 mg/kg bw/d of incomplete ossification of thoracic centrum (unchanged cartilage) and of tuberositas deltoidea (cartilage present), when

compared to concurrent control. However, the values were only slightly outside the historical control range. Incomplete ossification is a transient stage in pre – and postnatal development which is amended over time when the offspring gets more mature. As the underlying cartilages were entirely intact a complete recovery of the affected offspring is assumed. Therefore, those two findings might be regarded as treatment-related but not as adverse.

Applicant's summary and conclusion

Conclusions:
Pigment Yellow 155 causes neithr teratogenicity nor developmental toxicity in rats.
Executive summary:

In a prenatal developmental toxicity study, the test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the

expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Analyses confirmed the stability of the test substance in the vehicle over a period of 7 days. The results of the analyses of the test substance preparations of the high-dose group samples confirmed the correctness of the prepared concentrations or undercut the limits of the analytical method only marginally. Whereas the mean concentration values of the low- and mid-dose samples showed deviations below 80% of the nominal concentrations. The homogeneous distribution of the test substance in the vehicle (Tween 80 in drinking water (50 mg/100 mL)) was demonstrated in the high-dose group samples, but not in the samples of the low- and middose group. A comparison of the sample mass and volume (density) lead to the conclusion that an inclusion of air and hence a formation of foam took place while trying to extract a definitevolume of liquid sample with a syringe. However, the outcome of this study remained unaffected since the administered high-dose level was within or only marginally below the intended concentration.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 100, 300 and 1000 mg/kg bw/d Pigment Yellow 155 and controls.

Concerning thyroid hormone measurement, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, no treatment related findings were noted in the thyroid glands. All findings occurred either individually or were biologically equally distributed over control and´treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose.

There was an increase in incidence at 1000 mg/kg bw/d of incomplete ossification of thoraciccentrum (unchanged cartilage) and of tuberositas deltoidea (cartilage present), when compared to concurrent control. However, the values were only slightly outside the historical control range. Incomplete ossification is a transient stage in pre– and postnatal developmen which is amended over time when the offspring gets more mature. As the underlying cartilages were entirely intact a complete recovery of the affected offspring is assumed. Therefore, those two findings might be regarded as treatment-related but not as adverse.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Pigment Yellow 155 to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.