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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: ~ 11 weeks
- Housing: in groups of 5 during acclimatization, singly post-coitum
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one to one
- Proof of pregnancy: vaginal plug
Duration of treatment / exposure:
From Days 6 to 19 post-coitum, inclusive
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
900 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 28 d study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum

Food consumption Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the
reproductive organs
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
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
- The weight of each placenta (for live fetuses only).
Fetal examinations:
- External examinations: Yes: all per litter (each viable fetuses)
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine
intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Indices:
Pre-implantation loss (%) = [(number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100

Post-implantation loss (%) = [(number of implantation sites - number of live fetuses) / number of implantation sites] x 100

Viable fetuses affected/litter (%) = [number of viable fetuses affected or litter / number of viable fetuses/litter] x 100
Historical control data:
Study date range 2008 - 2012, No of studies 24

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals at 900 mg/kg bw/day had piloerection, salivation and pale feces. Lethargy was noted for several animals at this dose level, though it was mostly transient and/or occurred on only a few days of the treatment period. Restless behavior, flat or hunched posture, ptosis and uncoordinated
movements were also seen for females at this dose level. These occurred at a much lower incidence and were transient.

Salivation was noted for most females at 300 mg/kg bw/day, though this likely reflected a physiological response to the taste of the test substance and was not indicative of treatment related toxicity.

All other clinical signs seen for animals at 100 and 300 mg/kg bw/day including alopecia, pale feces, rales and piloerection occurred for 1-3 females in both groups. At the limited incidence observed, they were not were not considered to be toxicologically relevant. There were no clinical signs noted for control females.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was significantly lower for females at 900 mg/kg bw/day on Day 9 post coitum and absolute body weights and body weight gains were significantly lower than controls from Days 15-20 post coitum. On Day 20 post coitum, body weights were approximately 5% lower than controls. Female no. 78 had early resorptions only. While her low weights contributed to this difference, body weights and weight gains at 900 mg/kg bw/day remained significantly lower than controls even after discounting her data. Likewise, corrected maternal body weight gain was significantly decreased at 900 mg/kg bw/day (18.6 g versus 28.0 g in controls).

There was a significant difference in body weight gain between controls and animals treated at 900 mg/kg bw/day on Day 3 of post coitum. The difference from controls was only slight, and since this was before treatment commenced on Day 6, it was in no way treatment related.

Body weights and body weight gains were comparable to controls at 100 and 300 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 900 mg/kg bw/day absolute and relative food consumption were significantly lower than controls from Days 6-17 post coitum (relative food consumption was not significantly different Days 15-17).

Absolute and relative food consumption was significantly lower for females at 300 mg/kg bw/day Days 6-9 post coitum. The effect was only transient, not accompanied by changes in body weight, and was thus not considered toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant findings noted at the macroscopic examination.

Incidental findings noted for control and/or treated animals included pelvic dilation of the kidneys, dark red discoloration or dark red foci on the thymus, scabs and alopecia on various body regions. These were not treatment related or toxicologically relevant.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There were 20, 21, 21 and 21 pregnant females in the control, 100, 300 and 900 mg/kg bw/day groups, respectively with 20, 21, 21 and 20 litters available for evaluation.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
There were 20, 21, 21 and 21 pregnant females in the control, 100, 300 and 900 mg/kg bw/day groups, respectively with 20, 21, 21 and 20 litters available for evaluation. One female at 900 mg/kg bw/day, no. 78, had early resorptions only.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 900 mg/kg bw/day, no. 78, had early resorptions only.
Other effects:
no effects observed
Description (incidence and severity):
Placenta weights
There were no treatment related effects on placental weights up to 900 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 900 mg/kg bw/day, female fetal body weights were lower than controls. The difference from controls was only slight and the mean litter weights of most litters remained within the range of control fetal weights. Two litters were slightly lighter than the historical control range (dam no. 83: 2.6 g; dam no.
67: 2.8 g) were slightly lighter than the historical control range. The respective dams showed a very low corrected body weight gain (dam no. 83: -3.1 g; dam no. 67: +6.8g, compared to a group mean of +18.6 g), indicating these dams exhibited a considerably decreased growth. Therefore, the lower litter
weights observed in these dams were considered secondary to a reduction in maternal growth, and were not considered to be toxicologically relevant.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the fetal sex ratio in any group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment related effects on litter size seen up to 900 mg/kg bw/day.
The mean number of viable fetuses per litter was 12.0, 12.3, 12.2 and 11.7 in the control, 100, 300 and 900 mg/kg bw/day groups, respectively.
External malformations:
no effects observed
Description (incidence and severity):
No external malformations and developmental variations were observed in any of the fetuses, thus it was considered that test substance treatment at dose levels up to 900 mg/kg bw/day had no effect on fetal external morphology.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on fetal skeletal morphology. Examinations were done on 123(20), 129(21), 130(21) and 123(20) fetuses (litters) in the control, 100, 300 and 900 mg/kg bw/day groups, respectively.

Skeletal malformations were observed in 0(0), 4(4), 1(1) and 2(2) fetuses (litters) of the control, 100, 300 and 900 mg/kg bw/day groups, respectively. In the high dose group, the two malformations noted were severely malaligned sternebrae (fetus A084-02) and malpositioned metatarsals (fetus A070-14).
At 300 mg/kg bw/day, the affected fetus (no. A065-11) had malpositioned metacarpals (classified together with malpositioned metatarsals), which were also observed in one fetus at 100 mg/kg bw/day (no. A038-02). The three other fetuses in the low dose group with a skeletal malformation were nos.
A027-13, A031-13 (bent limb bones) and A025-01 (ectopic rib).
The skeletal malformations of this study occurred singly in either one or all dose groups, or twice in the low dose group. This distribution did not reveal a dose relationship and therefore the malformations were not considered to be treatment related.

Skeletal variations occurred in 45.7%, 45.7%, 46.0% and 41.9% of fetuses per litter in the control, 100, 300 and 900 mg/kg bw/day groups, respectively.
Among the variations a decreased mean litter proportion was observed for bent ribs at 100, 300 and 900 mg/kg bw/day (statistically significant in the mid and high dose groups). The mean litter incidences for this finding were 11.6%, 5.0%, 2.9% and 4.1% per litter in the control, 100, 300 and 900 mg/kg bw/day groups, respectively. Because no dose response for bent ribs could be established and because the mean litter incidence for this finding in all dose groups remained within the historical control data range (1.6-27.4% per litter), this variation was considered not to be treatment related.

Other skeletal variations observed in the test substance treated groups were 14th rudimentary rib, 14th full rib, 7th cervical rudimentary rib, 7th cervical full rib, slightly to moderately malaligned sternebrae, unossified sternebrae nos. 5 and/or 6, unossified sternebrae nos. 1,2,3 and/or 4, branched sternebra,
ossified vertebral centrum no. 1, reduced ossification of the skull, unossified hyoid, partially fused zygomatic arch, reduced ossification of vertebral centra, reduced ossification of vertebral arches and caudal shift of pelvic girdle. These variations were not considered to be treatment related, because
they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose relationship and/or occurred at frequencies within the historical control range.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on fetal visceral morphology.

One fetus with a visceral malformation was observed among the test substance treated groups. This fetus, no. A086-03 of the high dose group had an absent eye which was observed at cephalic examination. A similar finding, namely small eye, was observed at fresh visceral examination in control
fetus no. A007-03. Therefore, and taken into account the single occurrence, the absent eye was not considered to be caused by treatment.
Other visceral malformations affected control fetuses only. The fetus with the small eye (no. A007-03) also had transposition of the great vessels and a ventricular septum defect, and another control fetus (no. A018-11) had an appendix of the palate. Due to its occurrence in the control group, these
malformations were not related to treatment.

Visceral variations occurred in 11.4%, 11.7%, 10.3% and 14.3% of fetuses per litter in the control, 100, 300 and 900 mg/kg bw/day groups, respectively. Those observed in the test substance treated groups were small supernumerary liver lobe, liver appendix, discolored adrenal gland, convoluted ureter,
dilated ureter, pale spleen, partially undescended thymus horn and small thyroid. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose relationship and/or occurred at frequencies within the historical control range.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
900 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed.

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
visceral/soft tissue: eye
Description (incidence and severity):
One fetus with a visceral malformation was observed among the test substance treated groups. This fetus, no. A086-03 of the high dose group had an absent eye which was observed at cephalic examination. A similar finding, namely small eye, was observed at fresh visceral examination in control fetus no. A007-03. Therefore, and taken into account the single occurrence, the absent eye was not considered to be caused by treatment.
Other visceral malformations affected control fetuses only. The fetus with the small eye (no. A007-03) also had transposition of the great vessels and a ventricular septum defect, and another control fetus (no. A018-11) had an appendix of the palate. Due to its occurrence in the control group, these malformations were not related to treatment.

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
900 mg/kg bw/day (actual dose received)
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test item was established as being 300 mg/kg bw/day and developmental NOAEL was established as 900 mg/kg bw/day.
Executive summary:

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 100, 300 and 900 mg/kg bw/day (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, polyethylene glycol 400, alone. Females were checked daily for the presence of clinical signs. Body weights and food consumption of females were determined at periodic intervals. Formulations were prepared daily; formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

On Day 20 post-coitum, all animals were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations. Skeletal examinations were performed on approximately one-half of the fetuses. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Females at 900 mg/kg bw/day had treatment related clinical signs like piloerection and lethargy, among others, that occurred over several days but mostly resolved by the end of treatment. Females had lower body weights and weight gains from Days 15-20 post coitum and absolute and relative food consumption was also lower Days 6-17 post coitum. No maternal toxicity was observed in the 100 or 300 mg/kg bw/day groups. No developmental toxicity was observed in the 100, 300 and 900 mg/kg bw/day groups

Based on the results in this prenatal developmental toxicity study (OECD 414, conducted with GLP certification) the maternal No Observed Adverse Effect Level (NOAEL) for Irgacure 184 was established as being 300 mg/kg bw/day and developmental NOAEL was established as 900 mg/kg bw/day.