Registration Dossier

Administrative data

Description of key information

The key studies were conducted to recognised testing guidelines, and with GLP certification.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 February 2013 - 25 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted Oct. 2008
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
adopted May 2008
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Purity: 95.7 g/100g (by quanitative H-NMR; study No. 13L00016)
Homogeneity: given
Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Physical state/ appearance: liquid, viscous/ yellow, clear
Storage conditions: ambient (room temperature); under light exclusion
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species.
Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 41-43 days
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: groups of 5 in polysulfonate cages (floor area 2065cm²)
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal ad libitum
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance emulsions were prepared at least twice a week by stirring the appropriate amount in water containing 1% CMC. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer.

- Concentration in vehicle: 0.5, 1,5, 5.0g/100mL
- Volume used: 10ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was verified in the highest and lowest concentration. Correctness of concentration was confirmed in all concentrations at the beginning of the study. All measured values were in the range of 95 - 105%.

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The studies were carried out in compliance with the Principles of Good Laboratory Practice.
The stability of Lucirin TPO-L in water at room temperature for a period of 96 hours was demonstrated (project No. 01Y0459/038023; see PART III, Supplement).
Homogeneity of Lucirin TPO-L was verified in the highest and lowest concentration. Additionally, concentration control was verified in all concentrations at the beginning of the study.

See attached background material for more detail
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELCTION RATIONALE

In a test study 3 male and 3 female Wistar rats were treated with 1000mg/kg b.w. (BASF project No. 10C0459/03S008). The animals showed clear signs of systemic toxicity like poor general condition, piloerection, hunched posture and impaired body weight parameters. 2 females were sacrificed moribund on day 6. At scheduled necropsy on day 14, increased liver weights (absolute: males: 184%; females: 233% and relative: males: 221%; females: 223%), increased absolute (males: 111%; females: 140%) and relative (males: 135%; females: 134%) kidney weights, and increased adrenal gland and decreased spleen weights were observed in both genders.

Another set of male animals treated for 7 days with 500 mg/kg bw/d showed no clinical signs of concern, but liver weights were also increased, i.e. absolute (127%) and relative (121%) and spleen weights were decreased.
Based on these findings, 500mg/kg was chosen as the maximum dose level in the main study.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before administration as well as 2-5h after administration
- The following observations were included: mortality, clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at weekly intervals starting on day 0 prior to administration
- The following observations were recorded in a standard arena: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, appearance/consistency of feces, urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: weekly starting on day 0

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes (at weekly intervals over a period of one day)

WATER CONSUMPTION: Yes
- Time schedule for examinations: monitored daily by visual inspections of water bottles

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy (day 29)
- Animals fasted: Yes (at least for 16h, water available ad lib.)
- How many animals: all
- Parameters examined: Leucocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy (day 29)
- Animals fasted: Yes (at least for 16h, water available ad lib.)
- How many animals: all
- Parameters examined: ALT, AST, ALP, GGT, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: on day 23
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (no food or water provided)
- Parameters examined: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period (days 25/26)
- Dose groups that were examined: all
- Battery of functions tested: home cage observations in single animal cages, open field observations, sensory motor tests / reflexes, motor activity

OTHER:
- Oestrus cycle determination: vaginal smears were prepared in the morning for at least 3 weeks and at necropsy
- Sperm parameters: sperm motility, sperm morphology, sperm head count (cauda epididymis and testis)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Organ weights: anesthetized animals, adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles with coagulating glands, spleen, testes, thymus, thyroid glands, uterus with cervix

histopathologic examinations:
all animals: adrenal glands, left epididymis, kidneys, liver, spleen, stomach, testis

all high dose and control animals: bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, heart, ileum, jejunum, lung, lymph nodes, ovaries, peyer's patches, pituitary glands, prostate, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord, sternum, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

Gross lesions were examined in all animals affected.
Other examinations:
All observations made reported in attached background material
Statistics:
BODY WEIGHT, BODY WEIGHT CHANGE
A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
* for p ≤ 0.05
** for p ≤ 0.01
DUNNETT, C.W. (1955): A multiple comparison procedure for comparing several treatments with a control. JASA, Vol. 50, 1096-1121
DUNNETT, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, 482-491

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity, umber of cycles, cycle length
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians
* for p ≤ 0.05
** for p ≤ 0.01
SIEGEL, S. (1956): Non-parametric statistics for the behavioural sciences. McGraw-Hill New York
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in all high dose animals and most mid dose female animals shortly after treatment on several days throughout the study, starting on day 1. From the temporary, short appearance immediately after dosing it was concluded that salivation was induced by a bad taste of the test substance or local affection of the upper digestive tract.

(Tables IIA- 1 - IIA-24)
Mortality:
no mortality observed
Description (incidence):
No animals died prematurely.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in body weight or body weight gain were observed for male and female animal of test groups 1-3 (50, 150 and 500 mg/kg bw/d) when compared to control groups.

The significantly decreased body weight change value in female animals of test group 2 (150 mg/kg bw/d) on study day 7 was assessed as being incidental and not related to treatment.

(Tables IIA-27 - IIA-28)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (50, 150 and 500 mg/kg bw/d).

(Tables IIA-25 - IIA-26)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume. No overt changes in volume were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.

Regarding red blood cell parameters, in male animals of test group 3 (500 mg/kg bw/d) mean corpuscular volume (MCV) was lower, and in females of the same test group relative reticulocyte counts were higher compared to controls. However, both parameters were within historical control ranges (males: MCV 48.1-53.7 fL, females: relative reticulocyte counts 1.0-4.9%; PART III, Supplement). Therefore, these alterations were regarded as incidental and not treatment-related.

In male animals of test groups 1 and 3 (50 and 500 mg/kg bw/d) absolute monocyte counts were decreased, although not dose-dependently, and in males of test group 3 (500 mg/kg bw/d) relative monocyte counts were lower compared to controls. However, all medians were within historical control ranges (absolute monocyte counts 0.05-0.19 Giga/L; relative monocyte counts 0.9-2.5%; PART III, Supplement). Therefore, both changes were regarded as incidental and not treatment-related.

(Tables IB 1 – IB 2 Red blood cell and coagulation parameters)
(Tables IB 3 – IB 4 White blood cell parameters)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were
observed.
At the end of the administration period, in male animals of test group 1 (50 mg/kg bw/d) inorganic phosphate levels were increased. In female animals of test group 1 and 2 (50 and 150 mg/kg bw/d) creatinine levels were increased. However, both parameters were not changed dose-dependently and therefore the alterations were regarded as incidental and not treatment-related.


In males of test group 3 (500 mg/kg bw/d) cholesterol concentrations were decreased and in females of the same test group triglyceride and inorganic phosphate levels were increased. The inorganic phosphate levels in females were within the historical control range (phosphate 1.46-2.24 mmol/L; PART III, Supplement). The decreased cholesterol levels in males and increased triglyceride levels in females of test group 3 (500 mg/kg bw/d) were isolated in either sex and, therefore, they were regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002).

(Tables IB 5 – IB 6 Enzymes)
(Tables IB 7 – IB 8 Substrates)
(Tables IB 9 – IB 10 Electrolytes + minerals)
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed. Higher urobilinogen levels in the urine were measured in high dose rats of both sexes and, additionally, in mid dose males. Urobilinogen is synthesized in the intestine by bacteria and absorbed by the gut into the blood. With the blood circulation it normally enters the liver and is re-excreted via bile into the intestine. When a higher amount of urobilinogen is produced because of a greater breakdown of hemoglobin during hemolytic anemia, or a liver damage and cholestasis is preventing the biliary excretion of urobilinogen, higher amounts of the molecule are excreted via the kidneys.

However, in this study there was neither any indication of an anemia nor any sign of a liver cell damage or cholestasis. It is highly probable that the indicator field of the urobilinogen test strip is falsely affected by renal excreted compound or its metabolites coloring the urine light pastel orange. Therefore, because it was an isolated finding not accompanied by any red blood cell or liver cell alteration this change was regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002).

(Tables IB 11 – IB 14)
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean absolute and relative liver and kidney weights (in females only the relative kidney weight was increased) were significantly increased in high dose animals above current and historical control data (app. 140% for liver, 120% for kidney)

The liver and kidneys weight increases in males and the liver weight increase in females of test group 3 (500 mg/kg bw/d) were regarded as treatment-related. The liver weight increase in males (7.698 g) and females (4.864 g) of test group 2 (150 mg/kg bw/d) was within the range of the historical control data (males: 6.486-8.468 g; females: 4.194-5.426 g; PART III, Supplement). Therefore, the latter change was not considered to be treatment-related.

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

The liver weight increases in males and females of test group 3 (500 mg/kg bw/d) correlated with histopathological findings and were regarded as treatment-related. Although the kidney weight increase of males and females in test group 3 (500 mg/kg bw/d) had no histopathological correlate, it was regarded as treatment-related but not adverse. The liver weight increase in males (2.893%) and females (2.868%) of test group 2 (150 mg/kg bw/d) showed no histopathological correlate and were within the range of the historical controldata (males: 2.505-3.223%; females: 2.472-3.208%) and, therefore, were not considered to be treatment-related. All other mean relative weight parameters showed no significant differences when compared to the control group 0.

(Tables IC 1 – IC 8)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In test group 3 (500 mg/kg bw/d), 2 out of 5 males and 3 out of 5 females showed dark discoloration of the liver. However, no histopathological correlate was observed for this macroscopic finding. 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.

(Tables IC 9)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Correlating to the increased liver weights, minimal to slight centrilobular liver hypertrophy was observed in males (grade 2) and females (grade 1) of the high dose group. This finding was considered treatment related, but adaptive and not adverse. No histopathological correlate was found for the absolute and/or relative kidneys weight increase in males and females of this test group. However, this change was not considered to be adverse and most likely reflects an adaptive effect.

(Tables IC 10 – IC 13)
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Length and number of estrous cycle was unaffected.

(Table IA-45)

Sperm motility, sperm head counts, and incidence of abnormal sperms were unaffected by treatment.

(Tables IB 15)
Details on results:
Lucirin TPO-L was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg bw/d (test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 4 weeks.

Clinical examinations did not reveal treatment-related, adverse effects up to a dose level of 500 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of estrous cycles were obtained.
Salivation after treatment was seen in all animals of test group 3 (500 mg/kg bw/d) and sporadically in a few female animals of test group 2 (150 mg/kg bw/d). From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 500 mg/kg bw/d.

Regarding pathology, the absolute and/or relative weight increases observed in the liver and the kidneys of male and female animals of test group 3 (500 mg/kg bw/d) were regarded as treatment-related. In the liver, the weight increase correlated with centrilobular hepatocellular hypertrophy, affecting 5 out of 5 males (slight) and 4 out of 5 females (minimal). As the hepatocellular hypertrophy occurred without histopathological or clinical pathology alterations indicative of liver toxicity, the liver findings were considered to be treatment-related but adaptive and were therefore regarded as non-adverse (Hall et al., 2012). No histopathological correlate was found for the absolute and/or relative kidneys weight increase in males and females of test group 3 (500 mg/kg bw/d). However, this change was not considered to be adverse and most likely reflects an adaptive effect.

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.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
The oral administration of the test substance by gavage over a period of 4 weeks did not reveal signs of systemic toxicity in male and female Wistar rats up to a dose level of 500 mg/kg bw/d. In addition, no changes with regard to the reproductive organs were observed. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 500 mg/kg bw/d for male and female Wistar rats.
Executive summary:

METHODS

Lucirin TPO-L was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 4 weeks.

 

OBSERVATIONS

Food consumption and body weight were determined weekly. The animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out towards the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals, i.e. for at least 3 weeks an estrous cycle determination was performed and sperm parameters were examined after necropsy.

 

RESULTS

Analytics

The various analyses confirmed the stability of the test-substance preparations for a period of 96 hours at room temperature the homogeneous distribution of the test article in the vehicle, the correctness of the prepared concentrations.

 

Findings

The following test substance-related, relevant findings were noted:

 

Test group 3: 500 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology4

No treatment-related, adverse effects were observed.

 

Test group 2: 150 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

Test group 1: 50 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

CONCLUSION

The oral administration of Lucirin TPO-L by gavage over a period of 4 weeks did not reveal signs of systemic toxicity in male and female Wistar rats up to a dose level of 500 mg/kg bw/d. In addition, no changes with regard to the reproductive organs were observed. Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 500 mg/kg bw/d for male and female Wistar rats.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 2016- 27 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name of test substance: Irgacure TPO-L
CAS No.: 84434-11-7
Test substance No: 03/0459-3
Purity: 96.9% (study No.: 16L00101)
Homogeneity: Given (visually)
Physical state/ appearance: Liquid/ yellow
Storage conditions: Room temperature, under light exclusion
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Strain: Crl:WI(Han)
Supplier: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
Sex: Males/ females
Age when supplied: 36 ± 1 days
Age at the beginning of the administration period: 42 ± 1 days

Animal identification: Ear tattoo (animal number)

HOUSING AND DIET
The animals were housed together (5 animals per cage) in H-Temp polysulfonate cages type 2000P (floor area about 2065 cm2).

Motor activity measurements and FOB were conducted in polycarbonate cages type III (floor area about 800 cm2).

The cages and wire covers were supplied by TECNIPLAST, Hohenpeissenberg, Germany resp. by Ehret, Emmendingen, Germany. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH (Vienna, Austria) and large play tunnels (Art. 14153) supplied by PLEXX B.V. (Elst, Netherlands) were added for environmental enrichment.

The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C, a range of relative humidity of 30-70% and 15 air changes per hour. The day/night cycle was 12 hours (12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h). There were no or only minimal deviations from these limits.

The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls 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 (from water bottles) were available ad libitum.

On day of arrival, the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.

The test substance was administered daily for 13 weeks. Control animals received only the vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.
Route of administration:
oral: gavage
Details on route of administration:
The administration volume was 10 mL/kg body weight
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 1% Carboxymethyl cellulose was filled up to the desired volume,
subsequently mixed a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced twice a week. The administration volume was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE as a part of this study.

The stability of Irgacure TPO-L in double distilled water at room temperature for a period of 96 hours was proven before the start of the administration period (project No. 01Y0459/038023) with a comparable batch. Homogeneity was verified in 3 samples in the highest and lowest concentrations (was used as a concentration control at the same time) at the beginning of the administration period; additional concentration control analyses were verified in the intermediate concentrations. Moreover, homogeneity control analyses were verified in 3 samples in the highest and lowest concentrations (was used as a concentration control at the same time) at the end of the administration period; additional concentration control analyses were verified in 1 sample of the intermediate concentration.

Analytical methods
The methods used for analytical investigations of the test-substance preparations can be found in PART III (Supplement).- attached

Food analyses
The supplier assayed the food used in the study for chemical and microbiological contaminants.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

Bedding and enrichment analyses
The bedding and the enrichment are regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Duration of treatment / exposure:
The test substance was administered daily for 13 weeks. Control animals received only the vehicle.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
EXPERIMENTAL PROCEDURE
On day of arrival, the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
The test substance was administered daily for 13 weeks. Control animals received only the vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of food) of at least 16 hours.
Observations and examinations performed and frequency:
Mortality:
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations:
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

Detailed clinical observations:
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:

1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size

Food consumption:
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

Water consumption:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

Functional observational battery:
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the animals were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (Supplement).

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to:

1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:

1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait abnormalities
15. Activity/ arousal level
16. Feces excreted within 2 minutes (appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:

1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

Motor activity assessment:
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

Ophthalmoscopy:
Prior to the start of the administration period on day -4 the eyes of all animals and on study day 84 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).
Sacrifice and pathology:
After the administration period all animals were sacrificed and assessed by gross pathology.
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Male animal No. 21 of test group 2 (100 mg/kg bw/d), which died intercurrently, was necropsied and assessed by gross pathology as soon as possible after its death.
Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

Organ/tissue fixation

The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or
in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

The eyes of male animal No. 21, which died intercurrently, were fixed in 4% neutral-buffered
formaldehyde solution

Other examinations:
Please see below for tables on other examinations.
Statistics:
Please see below for statistics method tables.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in test groups 1 to 3 (20, 100 and 500 mg/kg bw/d).
Slight and moderate salivation shortly after treatment was observed in all male and female animals of test group 3 (500 mg/kg bw/d) and in 9 males and all female animals of test group 2 (100 mg/kg bw/d) on several days of the study. Additionally, 2 male animals of test group 3 (800 mg/kg bw/d) ploughed nose-first into bedding after application. From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both types of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract.

Semiclosed eyelid and labored respiration was observed after application in male animal No. 39 of test group 3 (500 mg/kg bw/d) on study day 88, only. The finding was assessed to be incidental.

A mass was palpable through skin in the right axillary region (>1.5 cm) of female animal No. 41 of test group 0 (0 mg/kg bw/d, control group) from study day 63 onwards. This finding was assed as spontaneous in nature.

(Tables IA 1 – IA 2)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Male animal No. 21 of test group 2 (100 mg/kg bw/d) was found dead on study day 75. No further animals died prematurely in the present study.

(Tables IA 1 – IA 2)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight values of male animals of test group 3 (500 mg/kg bw/d) were significantly lower from study day 42 onwards with a maximum of -18% on study day 84. The finding was assessed to be related to treatment and adverse. The same was true for body weight change values in male animals of test group 3, which were significantly lower from study day 35 onwards with a maximum by -30% on study day 84.

In female animals of test group 2 (100 mg /kg bw/d) the mean body weight values were decreased on study days 63, 70, 84 and 91 with a maximum by -6% on study day 91. In addition, the mean body weight change values were decreased on study days 70, 84 and 91
with a maximum by -12% on study day 91. As no dose-response relationship occurred the changes were assessed as being incidental and not related to treatment. The tables of the individual body weights are attached.

No significant changes of mean body weights and mean body weight change values were observed for male animals of test groups 1 and 2 (20 and 100 mg/kg bw/d) and for female animals of test groups 1 and 3 (20 and 500 mg/kg bw/d).

(Tables IA 7 – IA 14; figures 4.2.5.1. and 4.2.5.2.)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.

(Tables IA 3 – IA 6)
Food efficiency:
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.

(Tables IA 3 – IA 6)
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed.

All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.

(Tables IA 35 - IA 36)
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in rats of both sexes of test group 3 (500 mg/kg bw/d) hemoglobin, hematocrit, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. Additionally, in males of the same test group absolute reticulocyte counts were significantly increased. In females of test group 3 prothrombin time (Hepato Quick’s test, HQT) was significantly reduced. In males of this test group platelet counts were significantly higher compared to controls, but the values were within the historical control range (males, platelets 643-836 Giga/L). Therefore, this change was regarded as incidental and not treatment-related. The tables of the individual data are attached.

In females of test group 3 (500 mg/kg bw/d) total white blood cell (WBC) counts and absolute as well as relative lymphocyte counts were significantly increased and relative neutrophil counts were significantly decreased. Relative lymphocyte counts were already significantly higher in females of test group 2 (100 mg/kg bw/d), but this was the only changed differential blood cell count value and absolute lymphocyte counts were within the historical control range (females, absolute lymphocyte counts 1.85-3.35 Giga/L). Therefore, this isolated change in females of test group 2 was regarded as incidental and not treatment-related.

In males of test group 3 (500 mg/kg bw/d) absolute neutrophil counts were significantly increased. However, this was the only changed differential blood cell count in these individuals and total white blood cell counts were not changed and were within the historical
control range (males WBC 4.03-7.01 Giga/L). Therefore, the neutrophil count increase in males of test group 3 was regarded as maybe treatment-related but not adverse (ECETOC Technical Report No. 85, 2002). In males of test group 2 (100 mg/kg bw/d) absolute large unstained cell (LUCA) counts and relative monocyte counts were significantly decreased, but both parameters were not dose-dependently changed. Therefore, the changes in males of test group 2 were regarded as incidental and not treatment-related.

(Tables IB 1 – IB 2 Red blood cell and coagulation parameters)
(Tables IB 3 – IB 6 Total white and differential blood cell count)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period in rats of both sexes of test group 3 (500 mg/kg bw/d) alanine aminotransferase (ALT) activities, cholesterol, inorganic phosphate and calcium levels were significantly increased and glucose and chloride levels were significantly decreased. Additionally, in males of the same test group alkaline phosphatase (ALP) activity and potassium levels were significantly increased whereas in females of test group 3 albumin levels were significantly lower and triglyceride values were significantly higher compared to controls.

In males of test group 3 (500 mg/kg bw/d) urea and total bilirubin values were significantly higher and triglyceride values were significantly lower compared to controls. All mentioned values were within historical control ranges or only marginally below it (triglycerides)(males, urea 4.38-6.05 mmol/L; total bilirubin 0.56-2.76 µmol/L, triglycerides 0.61-1.29 mmol/L). In contrast to the changes in males of test group 3, in test group 2 (100 mg/kg bw/d) triglyceride values were significantly higher and total bilirubin values lower, but also in this test group the values were within the historical control ranges. In females of test group 3 (500 mg/kg bw/d) sodium levels were significantly lower compared to controls, but the mean was within the historical control range (females, sodium 139.8-145.4 mmol/L). In females of test group 1 (20 mg/kg bw/d) creatinine values were significantly increased, but this change was not dose-dependent. Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment-related.

(Tables IB 7 – IB 8 Enzymes)
(Tables IB 9 – IB 10 Substrates)
(Tables IB 11 – IB 12 Electrolytes + minerals)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In rats of both sexes of test group 3 (500 mg/kg bw/d) and, additionally, in females of test group 2 (100 mg/kg bw/d) urobilinogen levels in the urine were significantly increased.
Because higher urobilinogen levels were the only changed parameter in females of test group 2 and the red blood cell parameters and liver parameters in these individuals were not affected, this change in test group 2 was regarded as treatment-related but not adverse
(ECETOC Technical Report No. 85, 2002).

In females of test group 3 (500 mg/kg bw/d) urine bilirubin values and urine volume were significantly increased and specific gravity of the urine was significantly decreased. In males of the same test group ketone body levels in the urine as well as the counts of renal and
transitional epithelial cells in the urine sediment were significantly increased, whereas the urine pH value was significantly decreased.

(Tables IB 13 – IB 16)
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related findings were 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 with control group 0 (set to 100%), the following mean absolute weights were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold) (see table below).

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared with control group 0 (set to 100%), the following mean relative organ weights were significantly increased in one or more test groups (statistically significant changes printed in bold) (see below).

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significant decrease of the terminal body weight of males (315.52 g) was minimally below the historical control range (318.06 - 393.82 g) and was regarded as adverse. In females of test groups 2 and 3 (213.62 and 217.63 g), the significant decrease was within the historical control range (199.00 – 232.99 g) and showed no dose-dependency. Therefore, it was judged as not adverse. The statistical significant absolute and relative weight increases of the kidneys in males (abs. 2.869 g; rel. 0.91%) and females (abs. 2.022 g; rel. 0.93%) of test group 3 were above the respective historical control range values (males: abs. 2.010 - 2.488 g, rel. 0.551 – 0.671%; females: abs. 1.346 – 1.693 g, rel. 0.618 - 0.733 %) and were regarded as treatment-related. Although no clear histopathological correlate could be established, the magnitude of the weight increase was assumed to be adverse. The statistical significant relative weight increase of the kidneys in females of test group 2 (0.738%) was marginally above the maximum historical control value and had no histopathological correlate; therefore, it was regarded as not treatment-related. The statistical significant absolute and relative weight increases of the liver in males (abs. 10.855 g; rel. 3.428%) and females (abs. 8.627 g, rel. 3.965%) of test group 3 were above the historical control range values (males: abs. 6.835 – 9.261 g; rel. 2.063 – 2.39%; females: abs. 4.722 – 5.575 g; rel. 2.205 – 2.561%). These weight increases had a histopathological correlate and were considered treatment-related and adverse. In test group 2, the statistical significant relative weight increases of the liver in males (2.441%) was minimally above the historical control range. Although a histopathological correlate was noted, this was regarded as treatment-related but not adverse. In females of test group 2 the significant liver weight increase (2.522%) was within the historical control range and was regarded as incidental.

The statistical significant increase of the relative weight of the brain, epididymides, heart and testes in males of test group 3 were related secondary to the decreased terminal body weight and were not treatment-related.

(Tables IC 1 - IC 8)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In test group 3, the kidneys and livers of all male and female animals had a dark brown discoloration (except for the kidneys of one female). In 4 out of 10 males, liver and kidneys were also enlarged and the renal lymph nodes of 9 males had a dark brown discoloration.
Not all of these findings had a histopathological correlation but were regarded as treatment related.

Male animal No. 21 of test group 2 (100 mg/kg bw/d), which died intercurrently, showed a red-yellow mass of 8 mm in the skeletal muscle of the right side of the neck, accompanied by a coagulum in the skin of the same region and enlargement of the right axillary and
mediastinal lymph nodes. All of these findings were associated with a foreign body granuloma most likely due to a gavage error.

All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

(Tables IC 9 – IC 10)
Neuropathological findings:
no effects observed
Description (incidence and severity):
Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related
deviations to the control animals were noted for male and female animals of test groups 1-3
(20, 100 and 500 mg/kg bw/d).

For female animals of test group 3 (500 mg/kg bw/d) single interval No. 2 was significantly
decreased. This finding was assessed to be incidental and not related to treatment as only
one individual value was changed and the overall value did not differ significantly.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the kidneys and liver of male and females with incidences and grading according to the table below.

In test group 3, a minimal increase in the grading of cortical basophilic tubules was seen in males, whereas in females a slight increase in the incidence was noted. A fine granular gold-brown pigment storage was observed with H&E in the proximal convoluted tubules of 8 males and 3 females. Special stains performed exemplarily in the kidneys of selected animals for an approximate pigment identification showed following results below.

A minimal increase in the number and size of Schmorl-positive stained granules was noted in males and females of test group 3 compared with the control group. Based on this special stain, the gold-brown pigment seen with H&E in test group 3 might suggest a minimal treatment-related increase of lipofuscin. The minimal increase of the basophilic tubules and the minimal increase of a pigment storage in male and female animals of test group 3 were regarded as treatment-related. Although these findings are not consistent with the significant weight increase, in association with the strong significant relative weight increase (>25%), were assumed to be adverse.

Histopathology of the renal lymph nodes of test group 3 males revealed at H&E the presence of a gold-brown pigment in the sinus macrophages in 4 out of 9 animals, which correlated with hemosiderin in the Perl’s stain and was negative in the other special stains. The hemosiderin observed in these lymph nodes was not considered treatment-related, since its presence and amounts were within the normal pattern commonly found in lymph nodes of rats of this age and strain.

The hepatocyte hypertrophy in test group 3 was centrilobular in males and diffuse (with centrilobular accentuation) in females. It was regarded as treatment-related and adverse. The vacuolation of periportal hepatocytes, most probably a degeneration secondary to the diffuse hypertrophy, was also regarded as treatment-related and adverse. The centrilobular hypertrophy in males of test group 2 was considered treatment-related but not adverse, due to the lack of clinical chemical hepatic alterations.
A fine granular gold-brown pigment storage was observed with H&E diffusely distributed in hepatocytes and Kupffer cells of 6 males and 8 females in test group 3. Special stains performed exemplarily in the liver of selected animals for an approximate pigment identification showed following results:

The diffuse gold-brown pigment was positive in the Perl’s and Schmorl’s special stains and negative in the Hall’s stain, reflecting most likely the presence of hemosiderin and lipofuscin, respectively.

Male animal No. 21 of test group 2, which died intercurrently, showed a foreign body granuloma in the right side of the neck region and a thrombus in the surrounding area both containing vegetable fiber residues and bacteria. These findings were consistent with a gavage error which most likely caused the death of the animal. The enlargement of the right axillary and mediastinal lymph nodes, correlated with plasmocytosis in both lymph nodes and an additional mixed-cell inflammation and fibrosis in the right axillary lymph node. Furthermore, histopathology revealed plasmocytosis in the mandibular lymph nodes and an esophageal inflammation. All of these changes were considered to be secondary to the granuloma. A moderate myeloid extramedullary hematopoiesis in the spleen and a moderate myeloid hyperplasia in the bone marrow of this animal were most likely associated with the chronic inflammatory status of this animal.

All other histopathological 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.

(Tables IC11 – IC 16)
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
The following examinations were performed during FOB and have to be assessed individually:

Home cage observations:
(Males: table IA 15; females: table IA 22)

No test substance-related effects were observed.
A palpable mass through skin in axillary region on the right side (> 1.5 cm) was observed in female animal No. 41 of test group 0 (0 mg/kg bw/d, control group). This finding was assed as spontaneous in nature.

Open field observations:
(Males: tables IA 16 – IA 19; females: tables IA 23 – IA 26)

No test substance-related effects were observed.
A palpable mass through skin in axillary region on the right side (> 1.5 cm) was observed in female animal No. 41 of test group 0 (0 mg/kg bw/d, control group). This finding was assed as spontaneous in nature.
Slight salivation in female animal No. 77 of test group 3 (500 mg/kg bw/d) was observed during open field observation.

Sensorimotor tests/reflexes:
(Males: tables IA 20 – IA 21; females: tables IA 27 – IA 28)

No test substance-related effects were observed.
A palpable mass through skin in axillary region on the right side (> 1.5 cm) was observed in female animal No. 41 of test group 0 (0 mg/kg bw/d, control group). This finding was assed as spontaneous in nature.

Quantitative parameters:
(Males: table IA 29; females: table IA 30)

No test substance-related effects were observed.
Other effects:
no effects observed
Details on results:
Irgacure TPO-L was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 20 (test group 1), 100 (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 3 months.

With regard to clinical examinations, sings of systemic toxicity were observed in male animals of test group 3 (500 mg/kg bw/d), only, taking impaired body weight development into account.
No findings of toxicological concern were observed for female animals of test group 3 as well as for male and female animals of test groups 1 and 2 (20 and 100 mg/kg bw/d). Salivation after treatment was seen in several animals of test group 3 (500 mg/kg bw/d). In addition, 2 male animals of test group 3 ploughed nose-first into bedding after application. It was likely, that these findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. The findings were not considered to be adverse and toxicologically relevant effects.

Regarding clinical pathology decreased hemoglobin and hematocrit values in rats of both sexes of test group 3 (500 mg/kg bw/d) indicated an anemia. Higher urine urobilinogen levels confirmed hemolysis as primary cause. In males of this test group high absolute reticulocyte counts indicate still a regenerative nature of the anemia. However, decreased mean corpuscular volume (MCV), resulting in a decrease of mean corpuscular hemoglobin content (MCH)) were a sign of a dysregulation of the red blood cell synthesis, most probably due to an iron deficiency.
A second target organ in male and female rats of test group 3 (500 mg/kg bw/d) was the liver. Increased cholesterol levels in both sexes and higher triglyceride levels in females as well as decreased glucose levels in male and female rats as well as reduced prothrombin time and albumin levels in females were due to a dysregulated liver cell metabolism. Higher alanine aminotransferase (ALT) activities in rat of both sexes of test group 3 indicated a degradation of liver cell membranes. High alkaline phosphatase (ALP) activities in males of the mentioned test group and increased urine bilirubin levels in females were a consequence of liver cell swelling. The increased excretion of ketone bodies in the urine of males of test group 3 in combination with low serum glucose levels confirmed a katabolic metabolism in these individuals.
The following changes were most probably due to a partially compensated, metabolic acidosis in rats of both sexes of test group 3 (500 mg/kg bw/d): decreased serum chloride levels and increased serum calcium and inorganic phosphate levels in both sexes; increased serum potassium levels and low urine pH values in males.
The occurrence of renal epithelial cells in the urine sediment of male rats of test group 3 (500 mg/kg bw/d) were assessed as an indication of the affection of the tubule system in the kidneys.


Regarding pathology, target organs were the kidneys and liver in males and females. In test group 3 (500 mg/kg bw/d), the significant decrease of the terminal body weight of males (315.52 g) was minimally below the historical control range and was regarded as adverse. The significant absolute and relative kidneys weight increases in males (abs. 124%; rel. 154%) and females (abs. 133%; rel. 138%) were above the historical control values. Histopathology revealed in both sexes a minimal to slight increase of basophilic tubules and a minimal pigment storage increase (most likely lipofuscin) in the proximal convoluted tubules. Although these findings were not consistent with the significant weight increase, the strong relative weight increase of the kidneys per se (> 25% above the control groups) in both sexes, accompanied by the histopathological changes were altogether assessed as adverse.
In the liver, the hepatocyte hypertrophy (centrilobular in males and diffuse in females) was regarded as treatment-related and adverse in correlation with the strong absolute and relative liver weight increase (> 25% above the control groups) and with the clinical chemical hepatic alterations. In addition, a periportal vacuolation in females most probably secondary to the centrilobular hypertrophy, and a diffuse pigment storage in males and females (positive for hemosiderin and lipofuscin) were also assessed as treatment-related and adverse.
In test group 2 (100 mg/kg bw/d), the liver of males showed minimal statistical significant relative weight increases above the historical control range. This finding correlated with minimal centrilobular hypertrophy of hepatocytes. Since no clinical chemical alterations of the hepatic function were noted, these changes were regarded as treatment-related but not adverse.
In test group 1 (20 mg/kg bw/d), no treatment-related findings were observed. 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.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
urinalysis
Critical effects observed:
not specified

Absolute organ weights:

  Male animals Females
Test group (mg/kg bw/d) 1 (20) 2 (100) 3 (500) 1 (20) 2 (100) 3 (500)
Terminal body weight 99% 99% 81%** 100% 94%** 96%*
Kidneys 101% 106% 124%** 103% 103%** 133%**
Liver 103% 110% 127%** 98% 97%

155%**

*: p ≤0.05

**: p ≤0.01

Relative organ weights:

  Male animals Females
Test group (mg/kg bw/d) 1 (20) 2 (100) 3 (500) 1 (20) 2 (100) 3 (500)
Brain 102% 100% 124%**      
Epididymides 100% 104% 117%**      
Heart 101% 100% 113%**      
Kidneys 102% 107% 154%** 104% 110%* 138%**
Liver 104% 111%** 157%** 98% 103%** 161%**
Testes 104% 105% 126%**      

*: p ≤0.05

**: p ≤0.01

Kidneys:

   Male animals    Females  
Test group (mg/kg bw/d) 0 (0) 1 (20) 2 (100) 3 (500) 0 (0) 1 (20) 2 (100) 3 (500)
No. of animals 10 10 10 10 10 10 10 10
Tubules, basophilic 6 9 9 9 2 3 0 9
Grade 1 6 9 9 5 2 3   8
Grade 2       4       1
Pigment storage 0 1 0 8 0 0 0 3
Grade 1   1   5       2
Grade 2       3       1

  Perl's Hall's Schmorl's
Pigment Hemosiderin Bilirubin Lipofuscin
Test group 0 - - +
Test group 3 - - +

Liver:

   Male animals    Females  
Test group (mg/kg bw/d) 0 (0) 1 (20) 2 (100) 3 (500) 0 (0) 1 (20) 2 (100) 3 (500)
No. of animals 10 10 10 10 10 10 10 10
Hypertrophy, centrilobular 0 1 4 10 0 0 0 0
Grade 1   1 4          
Grade 2       5        
Grade 3       5        
Hypertrophy, diffuse 0 0 0 0 0 0 0 10
Grade 1               3
Grade 2               7
Vacuolation, periportal 0 0 0 1 0 0 0 5
Grade 1       1       4
Grade 2               1
Pigment storage, diffuse 0 0 0 6 0 0 0 8
Grade 1       6       5
Grade 2               3

  Perl's Hall's Schmorl's
Pigment Hemosiderin Bilirubin Lipofuscin
Test group 0 - - -
Test group 3 ++ - ++
Conclusions:
The oral administration of the test substance by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 500 mg/kg bw/d taking impaired body weight development, as well as altered clinical pathology parameters and pathology findings into account.

Therefore, under the conditions of the present study the NOAEL was 100 mg/kg bw/d for male and female Wistar rats.
Executive summary:

METHODS

Irgacure TPO-L was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 20 mg/kg bw/d (test group 1), 100 mg/kg bw/d (test group 2) and 500 mg/kg bw/d (test group 3) over a period of 3 months. Drinking water containing 1% Carboxymethyl cellulose served as vehicle. 

 

OBSERVATIONS

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.

Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.

 

RESULTS

Analytics

The various analyses confirmed:

the stability of the test-substance preparations for a period of 96 hours at room temperature,

the homogeneous distribution of the test substance in the vehicle,

the correctness of the prepared concentrations.

 

Findings

The following test substance-related, relevant findings were noted:

Test group 3: 500 mg/kg bw/d

Clinical Examinations

  • Significantly lower mean body weights in male animals from study day 42 onwards
  • Significantly decreased body weight changes in male animals from study day 35 onwards

  

Clinical Pathology

  • Decreased hemoglobin and hematocrit values, mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in both sexes
  • Increased absolute reticulocyte counts in males
  • Reduced prothrombin time in females
  • Increased total white blood cell (WBC), absolute and relative lymphocyte counts in females
  • Decreased relative neutrophil counts in females
  • Increased alanine aminotransferase (ALT) activities, cholesterol, inorganic phosphate and calcium levels in both sexes
  • Decreased glucose and chloride levels in both sexes
  • Increased alkaline phosphatase (ALP) activities and potassium levels in males
  • Increased triglyceride levels in females
  • Decreased albumin levels in females
  • Increased urine urobilinogen levels in both sexes
  • Increased ketone body levels and renal and transitional epithelial cell in the urine of males
  • Decreased urine pH value in males
  • Increased urine bilirubin levels and urine volume in females
  • Decreased urine specific gravity in females

Pathology

  • Significantly lower terminal body weight in male animals (-19%)
  • Significant liver weight increases in male (absolute: 127%; relative: 157%) and female (absolute: 155%, relative: 161%) animals
  • Significantly increased kidney weights in male (absolute: 124%; relative: 154%) and female (absolute: 133%, relative: 138%) animals
  • Centrilobular hypertrophy in the livers of all male (slight to moderate) and diffuse hypertrophy in the livers of female animals (minimal to slight)
  • Periportal vacuolation in the livers of one out of 10 male and 5 out of 10 female animals (minimal to slight)
  • Pigment storage (lipofuscin and hemosiderin) in the livers of 6 out of 10 male and 8 out of 10 female animals (minimal to slight)
  • Increase of basophilic tubules in the kidneys of 9 out of 10 males and 9 out of 10 females (minimal to slight)
  • Pigment storage (lipofuscin) in the kidneys of 8 out of 10 males and 3 out of 10 females

Test group 2: 100 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

Test group 1: 20 mg/kg bw/d

Clinical Examinations, Clinical Pathology and Pathology

No treatment-related, adverse effects were observed.

 

CONCLUSION

The oral administration of Irgacure TPO-L by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of systemic toxicity at a dose level of 500 mg/kg bw/d taking impaired body weight development, as well as altered clinical pathology parameters and pathology findings into account.

Therefore, under the conditions of the present study the NOAEL was 100 mg/kg bw/d for male and female Wistar rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

28 day repeated oral toxicity study:

Ethylphenyl(2,4,6 -trimethylbenzoyl) phosphinate was administered daily by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 50, 150, and 500 mg/kg bw for a period of 4 weeks. Special attention was paid to reproductive organs in male and female animals.

The doses were selected based on a screening study. Rats treated with 1000mg/kg b.w. showed clear signs of systemic toxicity like poor general condition, piloerection, hunched posture and impaired body weight parameters. 2 females were sacrificed moribund on day 6. Scheduled necropsy on day 14 revealed increased liver weights (absolute: males: 184%; females: 233% and relative: males: 221%; females: 223%), increased absolute (males: 111%; females: 140%) and relative (males: 135%; females: 134%) kidney weights, increased adrenal gland weights, and decreased spleen weights. Animals treated for 7 days with 500 mg/kg bw/d showed no clinical signs of concern, but liver weights were also increased (127%) and spleen weights were decreased. Based on these findings, 500mg/kg was chosen as the maximum dose level in the main study.

In the main study, clinical examinations did not reveal treatment-related, adverse effects up to a dose level of 500 mg/kg bw/d. In addition, no test substance-related effects on estrous cycle length and the number of estrous cycles were obtained. Salivation after treatment was seen in all high dose animals and sporadically in a few mid dose females. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. No treatment-related, adverse effects on clinical chemistry parameters and neurological functions were observed in any dose group. Regarding pathology, the absolute and/or relative weight increases observed in the liver (app. 140%) and the kidneys (app. 120%) of high dose male and female animals were regarded as treatment-related. In the liver, the weight increase correlated with centrilobular hepatocellular hypertrophy, affecting 5 out of 5 males (slight) and 4 out of 5 females (minimal). This finding was considered to be treatment-related but adaptive and was therefore regarded as non-adverse. No histopathological correlate was found for the absolute and/or relative kidneys weight increase. However, this change was not considered to be adverse and most likely reflects an adaptive effect. 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. Thus the no observed adverse effect level (NOAEL) was 500 mg/kg bw/d for male and female Wistar rats.

90 day subchronic oral toxicity study:

The test material was administered 10 male and 10 female rats by oral gavage daily at doses levels of 0, 10,20 100 and 500 mg/kg bw/d for a period of 90 days.

During the period of the study Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.

Ophthalmological examinations were performed before the beginning and at the end of the administration period. Beside this, a functional observational battery (FOB) as well as measurement of motor activity(MA) were carried out at the end of the administration period.

Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period all animals weresacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examination.

The various analyses of the dosing solutions confirmed the stability of the test-substance preparations for a period of 96 hours at roomtemperature, the homogeneous distribution of the test substance in the vehicle, the correctness of the prepared concentrations.

Test substance-related, adverse signs of systemic toxicity at a dose level of 500 mg/kg bw/d were observed taking impaired body weight development, as well as altered clinical pathology parameters and pathology findings into account.

Therefore, under the conditions of the present study the NOAEL was 100 mg/kg bw/d formale and female Wistar rats.

Justification for classification or non-classification

28 day study

The oral administration of ethyl phenyl (2,4,6 -trimethylbenzoyl)phosphinate by gavage over a period of 4 weeks did not reveal signs of systemic toxicity in male and female Wistar rats up to a dose level of 500 mg/kg bw/d. Thus no classification of the test substance for effects after repeated administration is required according to 67/548/EEC and CLP/EU-GHS.

90 day study

The oral administration of the test material by oral gavage over a period of 90 days did reveal treatment related effects of systmeic toxicity at the top does level of 500 mg/kg bw/d which included impaired body weight development, alter clinical chemistry pathology parameters and pathology findings. These effects were seen only in the highest dose level used and the NOEAL was determined to be 100mg/kg bw/d, this is above the guidance values of cited in Tables 3.9.2 and 3.9.3 of Regulation 1272/2008 (CLP regulation) that require the substance to be classified as a Catergory 1 or 2 toxicant, so based on the results of the study the test material does not require classifiaction for this end point.