Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2017 - 13 Nov 2017
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Batch Number 161005
Purity 96.9%
Date of production FEb 2016
Specific details on test material used for the study:
Name of test substance: Irgacure TPO-L
Test substance No.: 03/0459-3
Batch identification: 161005
CAS No.: 84434-11-7
Purity: 96.8 area-% (DB 1 capillary)
97.6 area-% (DB 1701 capillary)
(Final Report, Study Code: 16L00101)
Content: 94.6 g/100 g (Quantitative 1H-NMR spectroscopy)
94.4 g/100 g (Quantitative 31P-NMR spectroscopy)
(Final Report, Study Code: 16L00101)
Identity: Confirmed (Final Report, Study Code: 16L00101)
Homogeneity: Given
Storage stability: Expiry date: Feb 2019
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
The test facility is organizationally independent from the BASF SE sponsor division.

ADDITIONAL TEST SUBSTANCE INFORMATION

Chemical identity: Phosphinic acid, phenyl(2,4,6-trimethylbenzoyl)-, ethyl ester
Synonyms: Lucirin TPO - L
Physical state / appearance: Liquid / yellow
Storage conditions: Room temperature; under light exclusion

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
ANIMAL WELFARE
This study was performed in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.

Species and strain:
Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, at an age of about 10-12 weeks. Only animals free from clinical signs of disease were used for the investigations.

Animal identification:
The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm). After arrival of each cohort, the assignment of the animals to the different test groups was carried out by withdrawing them from the transport boxes and placing them into the cages at random. Each cohort was evenly distributed among the dose groups. After randomization the rats were identified consecutively and uniquely by ear tattoo.

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 strainhas been proven to be sensitive to substances with a teratogenic potential.

HOUSING AND DIET:
During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Ger-many (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of water and food consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals.

Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data).

For enrichment, wooden gnawing blocks were offered (Typ Lignocel® block large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany).

The animals were accommodated in fully air-conditioned rooms in which central air condition-ing maintained a range of temperature of 20-24°C and a range of relative humidity of 30-70%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study.

The day/night cycle was generally 12 hours (12 hours light from 06:00 h to 18:00 h and 12 hours darkness from 18:00 h to 06:00 h).

Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). The walls and the floor were cleaned once a week with water containing an appropriate disinfectant.

The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles) were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
Test substance preparations and preparation frequency:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.

For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
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 at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany.

The stability of the test substance in doubly distilled water at room temperature over a period of 96 hours had been verified before the start of the study in a similar batch (Project No.: 01Y0459/038023, see PART III). Based on the physico-chemical properties of Irgacure TPO-L, the stability of the substance preparation could be regarded as given in drinking water containing 0.5% Sodium carboxymethyl cellulose. Side reactions with Sodium carboxymethyl cellulose were unlikely.
Samples of the test substance preparations were sent to the analytical laboratory at the beginning of administration for verification of the concentrations. The samples were also used to verify the homogeneity of the low and the high concentrations (30 and 300 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

The body weight of the pregnant animals on day 0 varied between 147.1 – 190.4 g.

The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (0.5% Sodium carboxymethyl cellulose suspension in drinking water) in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined. The fetuses were removed from the uterus and investigated.
On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined according to 3.9.1. “Cesarean section” and 3.9.2. “Pathology”. The fetuses were removed from the uterus and investigated with the methods described in "Fetal examinations" section below.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.
Frequency of treatment:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.
Duration of test:
Gestation days 6-19
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
The aqueous test substance suspensions were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability.
The preparations were kept at room temperature.

For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% Sodium carboxymethyl cellulose suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.
Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).

The body weight of the pregnant animals on day 0 varied between 147.1 – 190.4 g.

The test substance preparations were administered to the animals once a day orally by gavage, rom implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (0.5% Sodium carboxymethyl cellulose suspension in drinking water) in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined according to Cesarean section and Pathology. The fetuses were removed from the uterus and investigated with the methods below.

Examinations

Maternal examinations:
Necropsy:
On GD 20 all surviving dams were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animal No. 86, which died intercurrently was sacrificed as soon as possible after its death and assessed by gross pathology.

Organ weights:
The following weights were determined in all animals sacrificed on schedule
1. Kidneys
2. Liver

The carcass weights (GROSSE-System) were transferred to the ACOPAT-System to calculate the relative organ weights.

Organ / Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
1. All gross lesions
2. Kidneys
3. Liver
4. Stomach

By mistake, the adrenal glands and the spleen were not weighted and fixed in formaldehyde solution as described in the study plan.

No further examinations or procedures were performed in the study.
Ovaries and uterine content:
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.

After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the following data were recorded:

- Weight of the unopened uterus

- Number of corpora lutea

- Number and distribution of implantation sites classified as:

• Live fetuses

• Dead implantations

a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)

b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)

c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

These data were used to calculate conception rate and pre- and postimplantation losses.

The conception rate (in %) was calculated according to the following formula:

conception rate (%) = (number of pregnant animals / number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:

preimplantation loss (%) = ((number of corpora lutea – number of implantations) / number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:

postimplantation loss (%) = ((number of implantations – number of live fetuses) / number of implantations) x 100
Fetal examinations:
Examinations of the fetuses after dissection from the uterus:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia.

Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus).

After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Evaluation criteria for assessing the fetuses:
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation:
A permanent structural change that is likely to adversely affect the survival or health.

Variation:
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.
The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
All fetal findings were listed in tables according to these classifications.
Statistics:
Yes see materials and methods below

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nearly all (22 out of 25) high-dose females (300 mg/kg bw/d) and one mid-dose female (100 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals shortly after treatment (i.e. within 2 hours post-dosing) and was initially observed on GD 7. The occurrence of salivation directly after dosing was assessed as treatment-related.

After 2 hours and up to 5 hours post-dosing no clinical signs or changes of general behavior were detected in any female of all test groups (30, 100 or 300 mg/kg bw/d), nor were any clinical signs observed before dosing in the morning.

See Part II, Tables IA-001 - IA-002 for individual observations.
Description (incidence and severity):
NA
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high-dose female (No. 86 - 300 mg/kg bw/d) was found dead after blood sampling on GD 20 without showing any preceding clinical signs of toxicity. The gross pathological exami-nation of this animal revealed no findings. The death after blood sampling was assessed as spontaneous and not treatment-related.
There were no further test substance-related or spontaneous mortalities in any females of all test groups (0, 30, 100 or 300 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight data:
The mean body weights and average body weight gain of the low-, mid- and high-dose dams (30, 100 or 300 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

See Part II, Tabs. IA-007 - IA-009 for individual observations.
See Fig. 4.2.1.5.1. for graphical representation.

Corrected (net) body weight gain:
The corrected body weight gain of test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

See Part II, Tables IA-010 for individual observations.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.

See Part II, Tables IA-005 - IA-006 for individual observations.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The mean water consumption of the dams in test group 3 (300 mg/kg bw/d) was statistically significantly increased on GD 10-13 and GD 17-20 (up to a maximum of 19% in comparison to the concurrent control). If calculated for the entire treatment or study periods, the high-dose dams consumed 10% or 9% more water than the controls. The finding was assessed as treatment-related.

The mean water consumption of the dams in test group 1 and 2 (30 and 100 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period. The statistically significantly decreased water consumption value in test group 2 on GD 15-17 is assessed as incidental.

See Part II, Tables IA-003 - IA-004 for individual observations.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20 in dams of test group 3 (300 mg/kg bw/d) red blood cell (RBC) counts, hemoglobin and hematocrit values were significantly decreased.

Additionally, in dams of test group 3 (300 mg/kg bw/d) relative neutrophil counts were significantly decreased and relative lymphocyte counts significantly increased. However, total white blood cell (WBC) counts and absolute neutrophil and lymphocyte counts were not altered. Absolute and relative lymphocyte counts in dams of test group 3 were slightly above and relative neutrophil counts slightly below historical control ranges, whereas WBC and absolute neutrophil counts in dams of this group were within historical control ranges (dams, relative neutrophils 31.3-42.3 %, relative lymphocytes 53.9-64.9 %, absolute neutrophils 1.53-2.28 Giga/L, absolute lymphocytes 2.41-3.94 Giga/L, WBC 4.47-6.24 Giga/L). Therefore, alterations of the lymphocyte counts were regarded as maybe treatment-related but not adverse, because this was the only changed differential blood cell fraction. Changes of the relative neutrophil cell counts were regarded as incidental and not treatment-related (ECETOC Technical Report No. 85, 2002).

In dams of test groups 2 and 3 (100 and 300 mg/kg bw/d) relative large unstained cell counts were significantly higher compared to controls, but the values were within the historical control range (dams, relative LUC 0.2-0.6 %). Therefore, this change was regarded as incidental and not treatment-related.

See Tables IB 1 Red blood cell parameters.
See Tables IB 2 - IB 3 Total white and differential blood cell count.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed. Individual clinical data is attached.

At gestation day 20, in dams of test group 3 (300 mg/kg bw/d) alanine aminotransferase (ALT) activities, urea and cholesterol levels were significantly higher compared to controls, whereas total bilirubin values were significantly decreased. Apart from cholesterol levels slightly above the historical control range, ALT activities, urea and total bilirubin levels were within their historical control ranges (dams, cholesterol 1.85-2.31 mmol/L, ALT 0.71-1.34 µkat/L, urea 4.52-6.70 mmol/L, total bilirubin 0.53-1.52 µmol/L). Therefore, the changes of ALT activities, urea and total bilirubin levels were regarded as incidental and not treatment-related. The cholesterol alteration in dams of test group 3 was regarded as treatment-related but not adverse, because only one of the measured clinical pathology liver parameters was changed (ECETOC Technical Report No 85, 2002; Hall et al., 2012).

See Table IB 4 Enzymes
See Tables IB 5 Substrates + minerals
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute organ weights:
When compared to control group 0 (set to 100%), the mean absolute weights of the livers were significantly increased in test group 3:

Absolute weights Females
Test group 1 2 3
(mg/kg bw/d) 30 100 300
Liver 99% 100% 110%**
* : p <= 0.05, **: p <= 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights:
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased in one or more test groups:

Relative weight Females
Test group 1 2 3
(mg/kg bw/d) 30 100 300
Liver 99% 100% 110%**
Kidneys 101% 102% 107%**
*: p <= 0.05, **: p <= 0.01

The slightly, but significantly increased absolute and relative liver weights of animals in test groups 3 lay above the range of historical control values. Therefore, a relation to treatment cannot be excluded.

The significantly increased relative weight of kidneys in test groups 3 was regarded as incidental, since the weight increase was only marginal and the relative weight parameters lay within the range of historical control values.

See Tables IC 1 – IC 2.

Uterus weight:
The mean gravid uterus weights of the animals of test groups 1-3 (30, 100 and 300 mg/kg
bw/d) were not influenced by the test substance. The differences between these groups and
the control group revealed no dose-dependency and were assessed to be without biological
relevance.

See Table IA-010.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross Lesions:
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.

See Table IC 3.

Pathology:
Summary tables of the results are to be found in the Part C of PART I; individual tables are to be found in Part C of PART II.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the control, mid- and high-dose groups (0, 100 and 300 mg/kg bw/d) and 100% in the low-dose group (30 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study (according to the test guidelines listed in chapter 2.3.).

There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 30, 100 and 300 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age;
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the control, mid- and high-dose groups (0, 100 and 300 mg/kg bw/d) and 100% in the low-dose group (30 mg/kg bw/d). With these rates, a sufficient
number of pregnant females were available for the purpose of the study (according to the test guidelines listed in chapter 2.3.).


There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 30, 100 and 300 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postim-plantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age;
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the control, mid- and high-dose groups (0, 100 and 300 mg/kg bw/d) and 100% in the low-dose group (30 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study (according to the test guidelines listed in chapter 2.3.).



There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 30, 100 and 300 mg/kg bw/d) in conception rate, in the mean number
of corpora lutea and implantation sites or in the values calculated for the pre- and the postim-plantation losses, the number of resorptions and viable fetuses. All observed differences are
considered to reflect the normal range of fluctuations for animals of this strain and age;

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
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical biochemistry

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.

See Table ID-001.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (30, 100 and 300 mg/kg bw/d) was comparable to the control fetuses.

See Table IA-013
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One external malformation was recorded for male control fetus No. 11-08, i.e. a cleft palate. This finding was associated with skeletal malformations. No external malformations occurred in treated animals. Total external malformations are summarised in the table below and presented in detail in attachments.

See Table ID-002 for fetal external examination results.
See Table ID-003 for detal external malformation results.

Fetal external variations:
No external variations were recorded.
See Table ID-004.

Fetal external unclassified observations:
One external unclassified observation, i.e. pointed lower jaw, was recorded in one male fetus of the control group (No. 11-08). This finding was confirmed during skeletal examination.
See Table ID-005.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examination of the fetuses:
See Table ID-012 for Skeletal examination of the fetuses.

Fetal skeletal malformations:
See Tables ID-013 - ID-016 for Fetal skeletal malformation observations.

Some skeletal malformations were detected in all test groups including the control (test groups 0-3; 0, 30, 100 and 300 mg/kg bw/d), as shown in Tab. 4.3.4.1.1. One control fetus had associated external findings. The incidences of these malformations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant (see summary table below).

‘Generalized disturbance of ossification’ was observed in one single fetus of test group 3. Since the finding was only observed in one individual case and no ontogenetic pattern was obvious, it was not assessed as treatment related.

The finding ‘cleft sternum’ was observed in one individual fetus of test groups 1 and 3 each. Cleft sternum can be found in higher incidences in the historical control data (affected fetuses/litter: 0.0 %, 0.0 – 1.0 %). The finding was not assessed as treatment-related.

‘Shortened humerus’ was observed in one fetus of test group 3, in two fetuses out of two litters of test group 2 and in one fetus of test group 1. The mean value of affected fetuses per litter of test groups 1 and 3 were within the historical control range (HCD, affected fetuses/litter: mean 0.2 %, 0.0 - 1.3 %). The mean value of test group 2 was slightly above the HCD. Since there was no relation to dose and it was only observed in individual fetuses, it was not assessed as treatment-related.

Fetal skeletal variations
See Tables ID-017 - ID-030 for Fetal skeletal variation observations.

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 dosing (see summary table below). The overall incidences of skeletal variations were comparable to the historical control data (PART III, SUPPLEMENT).

For a better overview, all skeletal variations with statistically significant differences between control and the treated groups were compiled in the table below (see summary table below). All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside historical control ranges were marked in bold and italics types.

The findings ‘incomplete ossification of parietal’ and ‘bipartite ossification of thoracic centrum’ were not related to dose and the mean values were clearly inside the historical control ranges. Therefore, both findings are not assessed as treatment-related.

The findings ‘incomplete ossification of skull’ and ‘unossified sternebra’ were statistically significantly increased in affected fetuses/litter in test group 3 (300 mg/kg bw/d). These values were outside the historical control range and assessed as treatment-related. The finding ‘wavy rib’ was dose-related, statistically significantly increased in affected fetuses/litter in test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d). The mean value of affected fetuses/litter of test group 2 was well within the historical control range and the increase may be incidental. The mean value of test group 3 was outside the historical control range and, therefore, assessed
as treatment-related.

Fetal skeletal unclassified cartilage observations:
See Tables ID-031 - ID-033 for Fetal skeletal unclassified cartilage observations.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups (see summary table below). 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. The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two fetuses each of the control group and test group 1 (30 mg/kg bw/d) had soft tissue malformations. These findings are not related to dosing and two of them (malpositioned kidney and abnormal lung lobation) can be found in the historical control data with comparable incidences, thus, they are not considered as treatment-related and adverse. The overall incidences of soft tissue malformations were comparable to those found in the historical control data (PART III, SUPPLEMENT - attached). Test groups 2 and 3 showed no malformations.

Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue malformations:
Two fetuses each of the control group and test group 1 (30 mg/kg bw/d) had soft tissue malformations. These findings are not related to dosing and two of them (malpositioned kidney and abnormal lung lobation) can be found in the historical control data with comparable incidences, thus, they are not considered as treatment-related and adverse.
The overall incidences of soft tissue malformations were comparable to those found in the historical control data (PART III, SUPPLEMENT). Test groups 2 and 3 showed no malformations.
See Tabs. ID-007 - ID-008 for Fetal soft tissue malformation observations.
See Tab. ID-006 for Soft tissue examination of the fetuses

Fetal soft tissue variations:
Three soft tissue variations were detected in all test groups including the control (0, 30, 100 or 300 mg/kg bw/d), i.e. short innominate, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All of them can be found in the historical control data at comparable incidences (PART III, SUPPLEMENT).- this is attached.

The finding ‘short innominate’ in two fetuses of two litters of test group 3 (affected fetuses/litter: 1.8 %) was in the range of the historical control data (HCD, affected fetuses/litter: mean 0.3 %, 0.0 -3.1 %) and, therefore, not assessed as treatment-related.

See Table ID-009 - ID-010 for Fetal soft tissue variations observations.

Fetal soft tissue unclassified observations:
No soft tissue unclassified observations were recorded.
See Table ID-011 for Fetal soft tissue unclassified observations.
Details on embryotoxic / teratogenic effects:
No differences of toxicological relevance between control and the treated groups 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 influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.



Regarding fetal examination, three variations - ‘incomplete ossification of skull’, ‘unossified sternebra’ and ‘wavy ribs’ - occurred at a dose of 300 mg/kg bw/d. They are common findings in rats and were observed in presence of maternal toxicity. They were assessed as minor effects indicating a developmental delay which is reversible. No unusual pattern of ossification was otherwise observable in the treated fetuses and the observed skeletal variations did not influence the overall rate of fetal variations (see Tab. 4.3.5.2. - this table has been attached) Therefore, these skeletal variations were assessed as treatment-related but not as adverse.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: prenatal developmental toxicity
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: prenatal developmental toxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes

Any other information on results incl. tables

Stability analysis:

The stability of the test substance preparations at room temperature over a period of 96 hours had been verified before the start of the study in a similar batch (Project No.: 01Y0459/038023, see PART III)

Homogeneity analysis of the test substance preparations:

The homogeneous distribution of the test substance in the vehicle (0.5% Sodium carboxymethyl cellulose in drinking water) was demonstrated (see PART III, SUPPLEMENT).

Concentration control analysis of the test substance preparations:

The results of the analysis of the aqueous test substance preparations confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical methods, i.e. were always above 90% and below 110% of the nominal concentrations (see PART III, Supplement).

Food analyses:

On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable. The EPA Fed. Reg. of 09 May 1979 (Vol. 44, No. 91, p. 27354) served as a guideline for maximum tolerable chemical contaminants. The amount of microorganisms did not exceed 1*105/g feed.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Drinking water analyses:

On the basis of the analytical findings, the drinking water was found to be suitable. The German Drinking Water Regulation (“Trinkwasserverordnung”) served as the guideline for maximum tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Bedding and enrichment analyses:

On the basis of the analytical findings, the bedding and the enrichment were found to be suitable. Levels given in Lab Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

EXAMINATION OF THE DAMS:

Summary tables are given in Part A of PART I. Individual values are given in Part A of PART II.

EXAMINATION OF THE FETUSES:

Summary tables are given in Part D of PART I. Individual values are given in Part D of PART II.

Total external malformations:

   Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d 
Test group 2
100 mg/kg bw/d 
Test group 3
300 mg/kg bw/d 
Litter  N  24 25 24 23
Fetuses  N  252 260 253 230
Fetal incidence N (%) 1 (0.4) 0 0 0

Litter incidence

N (%)

1 (4.2) 0 0 0

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

Total external unclassified observations:

   Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d 
Test group 2
100 mg/kg bw/d 
Test group 3
300 mg/kg bw/d 
Litter  N  24 25 24 23
Fetuses  N  252 260 253 230
Fetal incidence N (%) 1 (0.4) 0 0 0
Litter incidence N (%) 1 (4.2) 0 0 0
Affected fetuses/litter Mean% 0.4 0 0 0

Assessment of all fetal external, soft tissue and skeletal observations:

 See Table ID-034 Assessment of all fetal external, soft tissue and skeletal observations.

 External (Table ID-003), soft tissue (Tables ID-007 - ID-008) and skeletal (Tables ID-013 - ID-016) malformations were noted in all test groups (0, 30, 100 and 300 mg/kg bw/d). 

Six fetuses were multiple malformed: for male control fetus No. 11-08 a cleft palate was recorded combined with further skeletal findings (i.e. fused mandible, misshapen basisphenoid). Furthermore, in the same litter male fetus No. 11-05 showed a severely malformed vertebral column and/or ribs and malpositioned and bipartite sternebrae. Female control fetus No. 10-02 had a dextrocardia and an abnormal lung lobation, while male low-dose fetus No. 31-06 (30 mg/kg bw/d) had an abnormal lung lobation and a malpositioned adrenal. The findings in female mid-dose fetus No. 54-08 (100 mg/kg bw/d) consisted of a misshapen thoracic vertebra and malpositioned and bipartite sternebrae. Male mid-dose fetus No. 70-13 (100 mg/kg bw/d) had skeletal malformations concerning the upper limbs (i.e. shortened scapula, shortened humerus). No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of the high-dose group. All of them are present in the historical control data of the rat strain. Thus, a relationship of these six cases of malformed fetuses to the treatment is not assumed.

Other malformations, i.e. malpositioned kidney, misshapen tuberositas deltoidea, generalized disturbance of ossification, bipartite basisphenoid, and cleft sternum were not related to dose and nearly all of them can be found in the historical control data. An association of these findings to the treatment is also not assumed.

No external variations were recorded for any of the fetuses in this study. three soft tissue variations (Tabs. ID-009 - ID-010) and a range of skeletal variations (Tabs. ID-017 - ID-030) occurred in all test groups including the controls. None of the total incidences showed a relation to dosing (see summary table below). The majority of individual variations were equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.

The finding ‘wavy rib’ was dose-relatedly, statistically significantly increased in affected fetuses/litter in test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d). The mean value of affected fetuses/litter of test group 2 was well within the historical control range and the increase may be incidental. The mean value of test group 3 was outside the historical control range. The findings ‘incomplete ossification of skull’ and ‘unossified sternebra’ were statistically significantly increased in affected fetuses/litter in test group 3 (300 mg/kg bw/d). The mean values were outside the historical control range. 

Wavy ribs represent a common, minor finding in rat prenatal toxicity studies which is reversible. The underlying cause of the development of wavy ribs is probably an interference with the chondrification and ossification process of the ribs (Carney & Kimmel, 2007; Hofmann et al., 2016). 

Incomplete ossification of skull and unossified sternebra indicate a delay in ossification. Particularly, no changes in the underlying cartilages and no abnormalities in the respective bone structures were observed. Ossification of skull bones and sternebrae occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). In this study, maternal toxicity based on a normocytic-normochromic, anemic situation was observed at 300 mg/kg bw/d. 

In conclusion, all three variations are common findings in rats and were observed in presence of maternal toxicity at a dose of 300 mg/kg bw/d. They were assessed as minor effects indicating a developmental delay which is reversible. No unusual pattern of ossification was otherwise observable in the treated fetuses and the observed skeletal variations did not influence the overall rate of fetal variations (see summary table below) Therefore, these skeletal variations were assessed as treatment-related but not as adverse.

No unclassified soft tissue observations were recorded for any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external (Tab. ID-005) and the unclassified skeletal cartilage observations (Tabs. ID-031 - ID-033) which were observed in several fetuses of all test groups (0, 30, 100 and 300 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.

 

DISCUSSION:

In a prenatal developmental toxicity study the test substance Irgacure TPO-L 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 correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Neither clinical examinations nor determination of food consumption and body weights revealed any relevant difference between animals receiving 30, 100 or 300 mg/kg bw/d Irgacure TPO-L and the control. 

Nearly all females (22 out of 25) of the high-dose group (300 mg/kg bw/d) and one mid-dose female (100 mg/kg bw/d) showed transient salivation after treatment. This salivation occurred in the respective females shortly after each administration (within 2-hour post-dosing). The mean water consumption of the dams in test group 3 (300 mg/kg bw/d) was statistically significantly increased on GD 10-13 and GD 17-20 (up to a maximum of 19% in comparison to the concurrent control). The transient salivation after treatment of test group 3 and the increase in water consumption in test group 3 are considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. These findings are not considered to be a sign of systemic toxicity.

Regarding clinical pathology, in dams of test group 3 (300 mg/kg bw/d) decreased red blood cell (RBC) counts, hemoglobin and hematocrit values combined with no change of the red blood cell indices, mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV), indicated a normocytic-normochromic, anemic situation.

Regarding pathology, no treatment-related adverse changes were noted. Regarding the slightly, but significantly increased absolute and relative liver weights of animals in test group 3, a treatment-related adaptive change cannot be excluded. The weight parameters lay above the range of historical control values (see SUPPLEMENT). A histopathologic examination was not performed. All other 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 control and the treated groups 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 influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Regarding fetal examination, three variations - ‘incomplete ossification of skull’, ‘unossified sternebra’ and ‘wavy ribs’ - occurred at a dose of 300 mg/kg bw/d. They are common findings in rats and were observed in presence of maternal toxicity. They were assessed as minor effects indicating a developmental delay which is reversible. No unusual pattern of ossification was otherwise observable in the treated fetuses and the observed skeletal variations did not influence the overall rate of fetal variations (see summary table below) Therefore, these skeletal variations were assessed as treatment-related but not as adverse.

Individual fetal soft tissue malformations

Test group Dam No.-Fetus No., Sex  Finding 
0 (0 mg/kg bw/d)  10-02 F
16-06 M 
dextrocardia, abnormal lung lobation
malpositioned kidney
1 (30 mg/kg bw/d) 31-06 M
34-06 F 
abnormal lung lobation, malpositioned adrenal
malpositioned adrenal 
2 (100 mg/kg bw/d) None  
3 (300 mg/kg bw/d) None  

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

Total soft tissue malformations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 119 123 120 108
Fetal incidence  N (%) 2 (1.7) 2 (1.6) 0 0
Litter incidence N (%) 2 (8.3) 2 (8.0) 0 0
Affected fetuses/litter  Mean (%) 1.7 1.6 0 0

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

Total soft tissue variations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 119 123 120 108
Fetal incidence  N (%) 4 (3.4) 6 (4.9) 6 (5.0) 4 (3.7)
Litter incidence N (%) 4 (17) 4 (16) 4 (17) 4 (17)
Affected fetuses/litter  Mean (%) 3.1 5.3 5 3.8

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

Individual fetal skeletal malformations

Test group Dam No.-Fetus No., Sex  Finding 
0 (0 mg/kg bw/d) 3-06 M
5-09 F
11-05 M

11-08 M a)
misshapen tuberositas deltoidea
bipartite basisphenoid
severely malformed vertebral column and/or ribs malpositioned and bipartite sternebra
fused mandible, misshapen basisphenoid 
1 (30 mg/kg bw/d)  34-09 F
48-09 M 
cleft sternum
shortened humerus 
2 (100 mg/kg bw/d)  54-08 F
70-13 M
73-01 M
misshapen thoracic vertebra, malpositioned and bi-partite sternebra
shortened scapula, shortened humerus
shortened humerus 
3 (300 mg/kg bw/d) 77-03 F
77-09 M
 90-09 M 
cleft sternum
generalized disturbance of ossification
shortened humerus 

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

a) fetus with additional external malformation

Total skeletal malformations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 133 137 133 122
Fetal incidence  N (%) 4 (3.0) 2 (1.5) 3 (2.3) 3 (2.5)
Litter incidence N (%) 3 (13) 2 (8.0) 3 (13) 2 (8.7)
Affected fetuses/litter  Mean (%) 3.1 1.4 2.2 2.6

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

Total fetal skeletal variations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 133 137 133 122
Fetal incidence  N (%) 133 (100) 136 (99) 132 (99) 121 (99)
Litter incidence N (%) 24 (100) 25 (100) 24 (100) 23 (100)
Affected fetuses/litter  Mean (%) 100 99.3 99.4 99.1

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

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

Finding   Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
HCD
Mean %
(range) 
Incomplete ossification of parietal; unchanged cartilage 7.3 11.1 19.1** 9.3

1.04

(2.0-20.0)

Incomplete ossification of skull; unchanged cartilage 8.2 11.1 14.2 35.1**

8.1

(0.8 - 21.4)

Bipartite ossification of thoracic centrum; dumbbell-shaped cartilage of centrum  0 2.0* 0.7 0

0.5

(0.0 - 2.4)

Unossified sternebra; unchanged cartilage  2 2.5 2 13.6**

4.8

(0.0 - 10.3)

Wavy rib 3.1 3.2 9.7* 38.9**

4.9

(0.8 - 13.3)

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])

Total unclassified cartilage observations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 133 137 133 122
Fetal incidence  N (%) 99 (74) 91 (66) 92 (69) 93 (76)
Litter incidence N (%) 23 (96) 24 (96) 24 (100) 23 (100)
Affected fetuses/litter  Mean (%) 72 68 67.7 76.4

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

Total fetal malformations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 252 260 253 230
Fetal incidence  N (%) 6 (2.4) 4 (1.5) 3 (1.2) 3 (1.3)
Litter incidence N (%) 5 (21) 3 (12) 3 (13) 2 (8.7)
Affected fetuses/litter  Mean (%) 2.4 1.4 1.2 1.4

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

Total fetal variations

     Test group 0
0 mg/kg bw/d 
Test group 1
30 mg/kg bw/d
Test group 2
100 mg/kg bw/d
Test group 3
300 mg/kg bw/d 
Litter  N 24 25 24 23
Fetuses N 252 260 253 230
Fetal incidence  N (%) 137 (54) 142 (55) 138 (55) 125 (54)
Litter incidence N (%) 24 (100) 25 (100) 24 (100) 23 (100)
Affected fetuses/litter  Mean (%) 54.2 55.2 54.7 54.5

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

Applicant's summary and conclusion

Conclusions:
Discussion:
In a prenatal developmental toxicity study the test substance Irgacure TPO-L 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 correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

Neither clinical examinations nor determination of food consumption and body weights revealed any relevant difference between animals receiving 30, 100 or 300 mg/kg bw/d Irgacure TPO-L and the control.

Nearly all females (22 out of 25) of the high-dose group (300 mg/kg bw/d) and one mid-dose female (100 mg/kg bw/d) showed transient salivation after treatment. This salivation occurred in the respective females shortly after each administration (within 2-hour post-dosing). The mean water consumption of the dams in test group 3 (300 mg/kg bw/d) was statistically significantly increased on GD 10-13 and GD 17-20 (up to a maximum of 19% in comparison to the concurrent control). The transient salivation after treatment of test group 3 and the increase in water consumption in test group 3 are considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. These findings are not considered to be a sign of systemic toxicity.

Regarding clinical pathology, in dams of test group 3 (300 mg/kg bw/d) decreased red blood cell (RBC) counts, hemoglobin and hematocrit values combined with no change of the red blood cell indices, mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular volume (MCV), indicated a normocytic-normochromic, anemic situation.

Regarding pathology, no treatment-related adverse changes were noted. Regarding the slightly, but significantly increased absolute and relative liver weights of animals in test group 3, a treatment-related adaptive change cannot be excluded. The weight parameters lay above the range of historical control values (see SUPPLEMENT). A histopathologic examination was not performed. All other 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 control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corporalutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Regarding fetal examination, three variations - ‘incomplete ossification of skull’, ‘unossified sternebra’ and ‘wavy ribs’ - occurred at a dose of 300 mg/kg bw/d. They are common findings in rats and were observed in presence of maternal toxicity. They were assessed as minor effects indicating a developmental delay which is reversible. No unusual pattern of ossification was otherwise observable in the treated fetuses and the observed skeletal variations did not influence the overall rate of fetal variations (see Tab. 4.3.5.2.) Therefore, these skeletal variations were assessed as treatment-related but not as adverse.


Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of Irgacure TPO-L to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused adverse effects at the highest tested dose of 300 mg/kg bw/d.

Maternal toxicity was determined by a normocytic-normochromic, anemic situation. Increases in absolute and relative liver weights indicated a treatment-related adaptive change. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/d.

In fetuses, three skeletal variations affecting the skull, ribs and sternebrae were assessed as treatment-related but not as adverse. These minor and reversible effects indicate a developmental delay which is reversible. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg bw/d while the no observed effect level (NOEL) for prenatal developmental toxicity is 100 mg/kg bw/d.
Executive summary:

Irgacure TPO-L was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistarrats by gavage at doses of 30, 100 and 300 mg/kg body weight/day (mg/kg bw/d) on gestationdays (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose suspension in drinking water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites.

OBSERVATIONS

Water consumption, food consumption and body weights of the animals were recorded regu-larly throughout the study period. The state of health of the animals was checked each day.

On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation

(under isoflurane anesthesia) and assessed by gross pathology (including weight determina-tions of the kidneys, liver, unopened uterus and placentas). For each dam, corpora lutea werecounted and number and distribution of implantation sites (differentiated between resorptions,live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed,

weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined forfor soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

Analytics

• The stability of the test substance in doubly distilled water at room temperature was demon-strated for a period of 96 hours in a similar batch.

• The homogeneous distribution of the test substance in the vehicle (0.5% Sodiumcarboxymethyl cellulose suspension in drinking water) was confirmed.

• The correctness of the prepared concentrations was shown.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3 (300 mg/kg bw/d):

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in dams.

• No test substance-related adverse effects on gestational parameters or fetuses.

Test group 2 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses.

Test group 1 (30 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses.