Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
based on EPA-TSCA Guideline "Functional Observational Battery" & EPA-TSCA Guideline " Neuropathology" (Federal Register Vol. 50, No. 188)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr Karl Thomae GmbH, Biberach/Riss, FRG
- Age at study initiation: app. 5 weeks
- Weight at study initiation: 191 (177 - 210) g for males, 156 (143 - 217) g for females
- Housing: single in DK III stainless steel wire cages. The animal cages were placed on the racks in a manner such that uniform test conditions (air inflow/air outflow/light) were guaranteed
- Diet: ground Kliba rat/mouse/hamster maintenance laboratory diet, GLP 343 meal, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod: 12 hrs dark /12 hrs light
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparations were made prior to each administration.

VEHICLE
- Concentration in vehicle:
_________________________________
Dose Dose Concentration
group (mg/kg bw) (g/100 ml)
---------------------------------
0 0 -
1 100 1.0
3 300 3.0
4 1000 10
_______________________________
- Amount of vehicle (if gavage): 10 ml/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in the vehicle was verified for a period of 24h.
The concentrations from the beginning and the end of the study were verified via UV spectroscopy at 379 nm and found to be within 10% of the target concentration.
Duration of treatment / exposure:
3 months (total of 64 applications)
Frequency of treatment:
once daily on workdays (5days/week, excepted for 3 working days over the whole experimental period)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of a test study where a dose of 1000 mg/kg bw/day was applied 10 times in 14 days and did not lead to marked toxicity besides a minimal reduction in body weight (6 %) in male but not in female animals if compared to controls.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for any evident signs of toxicity / mortality; once weekly for exact clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, once a week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning on days 33 and 87 after the beginning of administration
- Animals fasted: No
- How many animals: all
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above, HAEMATOLOGY
- Parameters examined: 1) enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase: 2) blood chemistry (sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once prior to the start of the study (Oct 16 1987), at 1, 6, and 24 hours after the first administration and before the daily exposure on days 7 (Oct 26 1987) and 14 (Nov 2 1987) and then monthly to day 91 (Nov 30 1987; Dec 21 1987 and Jan 18 1988)
- Dose groups that were examined: all animals
- Battery of functions tested:
- observation of present/absent:
tremors, convulsions, lacrimation/secretion of pigmented tears, salivation, piloerection, pupil size (constriction/dilatation), diarrhea, vocalization, paresis, paralysis and staxia
- Appearance of impairments refering to:
general appearance, posture, skin colour, locomotor activity, respiration, urination (volume, odour, colour), pupillary reflex, winking reflex, righting reflex, behaviour, grip strength, body tone, vision, olfaction, audition, sensitivity of the body surface, pain perception (hot plate), tail Pinch, toe Pinch, visual placing response.
Sacrifice and pathology:
1) Perfusion fixation
Organs of the first three animals per group and sex were fixed by perfusion using 2.5% glutaraldehyde and 4% formaldehyde.

GROSS PATHOLOGY: Yes: brain, gasserian ganglia of both sides, dorsal root ganglia (C3-C6 and L1-L4 ), proximal sciatic nerve (mid thigh and sciatic notch ), gastrocnemius muscle, kidneys, adrenal glands, skin, medulla oblongata, spinal cord at cervical ( C3-C6) and lumbar swelling (L1-L4 ), dorsal and ventral root fibers (C3-C6 and L1-L4 ), sural and tibial nerve (knee region), liver, spleen, testes, all gross lesions

HISTOPATHOLOGY: Yes
all groups: gastrocnemius muscle, skin ( auricle), skin ( paw region), liver, kidneys, testes, spleen, all gross lesions (if observed), gasserian ganglia, dorsal root ganglia, dorsal and ventral root fibres, proximal sciatic nerve, sural and tibial nerve
high dose and control: brain (forebrain, cerebrum, midbrain, cerebellum, pons), medulla oblongata, spinal cord at C3-C6 and at L1-L4


2) Immersion fixation
Organs of the remaining 7 animals per group and sex were necropsied and assessed by gross pathology prior to fixation in 4% formaldehyde solution.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
all groups: liver, kidney, spleen, adrenals, testes, left hind extermity with all skeletal muscles and sciativ nerve roots, all gross lesions
high dose and control: gasserian ganglia of both sides, spinal cord at cervical and lumbar swelling (C3-C6 and L1-L4), dorsal root ganglia (C3-C6 and L1-L4 ), dorsal and ventral root fibers (C3-C6 and L1-L4 ), proximal sciatic nerve (mid thigh and sciatic notch), sural and tibial nerve (knee region), brain, medulla oblongata,
Statistics:
The statistical assessement of the findings was based on the analysis of variance (ANOVA) followed by a Dunnett´s test (Dunnett CW, J. Amer. Statist. Assoc., 50, 1096-1121, 1955,´Dunnett CW, Biometrics, 20, 482-491, 1964.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The females of the 1000 mg/kg group showed a reduced general state of health. Lesions on the hairless skin of the extremities and reddening and scale formation on the ears were reported for males and females of the 1000 mg/kg group. In males of both, the 300 and the 1000 mg/kg group, the testes were reduced in size, which was palpable from week 6 onwards.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females of the 1000 mg/kg group died during the experimental period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For males and females of the 1000 mg/kg group body weight was reduced (23% in males, 8% in females). At 300 mg/kg, body weight reduction only concerned the males (10%). Body weight gain was also reduced by 38% (high dose males), 16% (mid dose males), and 16% (high dose females).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
An increase in food consumption was reported for the females of the 1000 mg/kg.

Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the females of the 1000 mg/kg group, erythrocytes, hemoglobin, hematocrit and thromboplastin time (Hepato Quick test) were decreased. Hemoglobin and hematocrit values were also found to be reduced in females exposed to 300 mg/kg. In contrast, leucocytes, platelets, eosinophilic granulocytes, neutrophilic polymorphonuclears, and calcium levels were increased in mid and high dose females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum alkaline phosphatase and gamma-glutamyltransferase activity were significantly increased in both sexes of the high dose group (1000mg/kg). In addition, alanine aminotransferase activity was significantly increased in high dose males. Triglyceride levels were decreased in both sexes of the high dose group, while cholestrol levels were increased and clotting time was decreased in females only. These findings are attributed to liver dysfunction.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed increased absolute and relative kidney and liver weights in the females and increased relative kidney and liver weight in males of the 1000 mg/kg group and females of the 300mg/kg group. In all males in the mid and high dose group, the absolute and relative testes weights were decreased, on average by about 50%.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Neither functional defects nor any other signs of neurotoxicity were observed.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Despite altered blood and urine parameters and increased liver and kidney weight, histopathology revealed no damage on these organs. In the testes of mid and high dose males marked diffuse atrophy of the testicular parenchyma and a slight to moderate interstitial oedema was detected. No further substance related effects were found. Especially, no damage was detected in any samples taken from nervous tissues.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
Critical effects observed:
not specified

Body ans testes weight development; results of testes patology:

Dose group/finding

0

mg/kg bw/day

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

Body weight

day 0

190.4 ± 7.9

190.1 ± 7.4

190.9 ± 5.1

192.4 ± 5.5

day 91

498.8 ± 44

493.5 ± 29.8

451.3 ± 37

384.0 ± 27.6

Testes

10

10

10

10

absolute weights

3.563 ± 0.193 g

3.68 ± 0.353 g

1.691 ± 0.328 g

1.693 ± 0.369 g

relative weights

0.78 ± 0.062 g

0.818 ± 0.094 g

0.421 ± 0.1 g

0.477 ± 0.1 g

diffuse atrophy

--

--

10/10

10/10

edema

--

--

10/10

10/10

focal atrophy

2/10

1/10

--

--

vacuolar degeneration

7/10

10/10

--

--

reduced spermiogenesis

--

1/10

--

--

Incidence and grading of microscopic findings:

Dose group

Grading

0

mg/kg bw/day

100

mg/kg bw/day

300

mg/kg bw/day

1000

mg/kg bw/day

Testes

10

10

10

10

diffuse atrophy

--

--

10

10

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

--

--

1

--

4 (marked)

--

--

9

10

5 (severe)

--

--

--

--

edema

--

--

10

10

1 (minimal)

--

--

--

--

2 (slight)

--

--

3

7

3 (moderate)

--

--

7

3

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

focal atrophy

2

1

--

--

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

1

--

--

--

4 (marked)

1

1

--

--

5 (severe)

--

--

--

--

vacuolar degeneration

7

10

--

--

1 (minimal)

3

3

--

--

2 (slight)

1

6

--

--

3 (moderate)

3

1

--

--

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

reduced spermiogenesis

--

--

1

--

--

1 (minimal)

--

--

--

--

2 (slight)

--

--

--

--

3 (moderate)

--

1

--

--

4 (marked)

--

--

--

--

5 (severe)

--

--

--

--

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare (M .H .W .) guidelines 1986 for a twenty-eight day repeat dose oral toxicity study
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Interfauna (UK) Limited, Wyton, Huntingdon, Cambridgeshire
- Age at study initiation: 6-7 weeks old. Only animals free from clinical signs of disease were used for the study.
- Weight at study initiation: 152 - 192 g for males, 155 - 189 g for females
- Housing: groups of 5 by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet, ad libitum: SQC Rat and Mouse pelleted diet No 1 Expanded, Special Diet Services Limited, Witham, Essex, UK. The animals were allowed free access to food, except during urine collection when food was withdrawn overnight.
- Water, ad libitum: tap water ad lib.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24°C
- Humidity (%): 30 - 67 % (on several occasions during the study the humidity fell slightly below the protocol limit (40%) but this was considered not to affect the purpose or integrity of the study)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 hours (6.00 am - 600 pm/ 6.00 pm - 6.00 am)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly and stored in the dark at 4°C.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is hardly soluble in water. Administration via diet is impossible due to palatability reasons.
- Amount of vehicle (if gavage): 4 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material formulation for at least nine days was determined by Safepharm Analytical Laboratory prior to the start of the study. Since no differences were detected in concentration from samples taken from the top, the middle, or the bottom, the formulation was homogenous. Concentrations were checked at weekly intervals, i.e. for every new formulation. All results were between 91% and 109% of the target concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 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 selection rationale: In a pretest study the test substance was administered to groups of 3 male and 3 female rats at a dose level of 20, 250, 500, 750 and 1000 mg/kg bw/day for 14 days. Body weight, clinical signs and macroscopic findings were recorded. Animals of the highest dose group were killed in extremis. Whereas at 750 and 500mg/kg b.w. adverse clinical signs and reduction in body weight gain was detected, this was reduced to minor adverse clinical signs at 250 mg/kg b.w.. No abnormalities were observed in the control group and at 50 mg/kg b.w.
- satellite groups: 5 male and 5 female animals of the control and the high dose group
- Rationale for selecting satellite groups: recovery examination
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no
Observations and examinations performed and frequency:
OBSERVATION FOR CLINICAL SIGNS: Yes
- Time schedule: immediately before dosing and one and five hours on weekdays, before dosing and one hour after dosing on weekends and public holidays. During the treatment-free period satellite group animals were observed twice daily, morning and afternoon (once daily on weekends).
- Parameters: overt signs of toxicity, ill-health, behavioural changes

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on the day before the start of treatment (day 0) and on days 7, 14, 21 and 28 and in the case of satellite group animals on days 35 and 44. Body weights were also recorded at necropsy

FOOD CONSUMPTION: Yes
- Food consumption: weekly

WATER CONSUMPTION : Yes
- Time schedule for examinations: water intake was observed daily by visual inspection of the water bottles per group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period and in satellite group animals at the end of the treatment-free period (blood samples were obtained by orbital sinus puncture)
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: No
- How many animals: all animals
- Parameters examined: haematology (heamatocrit (Hct), haemoglobin (HB), erythrocyte cout (RBC), total leucocyte count (WBC), erythrocyte indices [mean cell haemoglobin (MCH), mean cell volume (MCV) and mean cell haemoglobin concentration (MCHC)]); blood chemistry (blood urea, total protein, albumin, albumin/globulin ratio [by calculation], sodium, potassium, chloride, calcium, inorganic phosphorus, creatinine, alkaline phosphatase [AP], alanine aminotransferase [ALAT], aspartate aminotransferase [ASAT], glucose, gamma glutamyl transpeptidase (γGT), triglycerides, total cholesterol and bilirubin)
In satellite group animals only those parameters were examined, that showed possible treatment-related changes after 28 days.

URINALYSIS: Yes
- Time schedule for collection of urine: urinalysis was performed on all animals within the week prior to necropsy (week 4 or week 6 (satellite group))
- Metabolism cages used for collection of urine: Yes, urine was collected over a period of 16 hours.
- Animals fasted: Yes, the animals were maintained under conditions of normal hydration during collection, but without access to food.
- Parameters examined: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, reducing substances, blood

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, the following organs, dissected free from fat, were weighed before fixation: adrenals, brain, gonads, heart, kidneys, liver, pituitary and spleen

HISTOPATHOLOGY: Yes, Samples were removed from all animals and preserved in 10% buffered formalin
All animals were assessed by gross pathology, followed by histopathological examinations: adrenals, aorta (thoracic), bone and bone marrow (femur), brain, caecum, colon, duodenum, eyes, gross lesions, heart, muscle (skeletal), pancreas, pituitary, rectum, sciatic nerve, skin (hind limb), spleen, stomach, testes, thymus, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), thyroid/parathyroid, trachea, urinary bladder, oesophagus and ovaries. The following preserved tissues from all high dose and control group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm and stained with haematoxylin and eosin: adrenals, heart, kidneys, liver, spleen and testes. Macroscopically observed lesions were also processed in addition to the above. Since there were indications of treatment-related changes in the liver, kidneys and testes, examination of these tissues was subsequently extended to the remaining groups.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogenous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test. Probability values were calculated as follows: p < 0.001, ***; p < 0.01, **; p < 0.05, * and p > 0.05, not significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
750mg/kg: The following clinical signs were observed from day three onward: increased salivation, red/brown staining around the snout and mouth, wet fur, red/brown staining of the fur, hair loss, pilo-erection, hunched posture, lethargy, ptosis, diuresis, diarrhoea and abdominal distension, single incidence of vocalisation
Satellite high dose animals recovered immediately following cessation of dasing and appeared normal throughout the treatment-free period .

250mg/kg: Animals from this group showed the same clinical signs, though with less severity, from day four onward, which were observed in the high dose group, with the exception of diarrhoea, abdominal distension, and vocalisation.

50mg/kg: no clinical signs were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female from the satellite high dose group was found dead on day four and one female from the satellite control group died during blood sampling on day forty-two .
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
750mg/kg: Bodyweight and bodyweight gains were significantly reduced in week four in males and females. Four females lost bodyweight within this week. Females of the satellite group were not affected. Weight gains quickly recovered in satellite high dose males during the treatment-free period. Reduced weight gain was also observed in high dose males throughout weeks 1 and 2, while an increase in bodyweight gain was observed in females in week 3.

250mg/kg: Bodyweight gains were reduced in males and females throughout week 4.

No adverse effect on bodyweight was noted in animals treated with 50 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in food consumption were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
High dose animals of both sexes and intermediate dose females showed reduced haemoglobin levels. Calculation of erythrocyte indices showed reductions in mean corpuscular volume and mean corpuscular haemoglobin. Platelet counts were also slightly reduced in high dose females but clotting potential (by 'Hepato-Quick') was unaffected. Most values for the above parameters were within the normal range, but occasionally individual or mean values were slightly outside the normal range. These minor changes were probably associated with the general condition of the animals rather than being direct haematopathological effects.

Males treated with 750 mg/kg/day showed a statistically significant increase in leucocyte counts compared with controls (p =<0 .001) and a slight increase was also noted in intermediate dose males. The increase was predominantly in the lymphocyte fraction and a slight increase in lymphocyte counts was also apparent for high and intermediate dose females. Lymphocyte and total leucocyte counts for individual animals were all entirely within the normal range for rats of this strain and age. However, a slight increase in neutrophil counts involved one value outside the normal range. Neither lymphocytosis nor granulocytosis have been assnciated with specific chemical insults and, in the absence of any other evidence of infection, the intergroup differences were considered to be entirely fortuitous.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Toxicologically significant blood chemical changes were observed, indicative of cholestatic hepatic injury and renal obstruction.

Animals of both sexes treated with 750 mg/kg/day showed marked elevations in cholesterol and gamma glutamyl transpeptidase with males also showing increased alkaline phosphatase. Bilirubin was increased in high dose animals of both sexes and in mid-dose females.
Creatinine levels were substantially elevated in males and females treated with 750 mg/kg/day with some individual animals showing extremely high values. This change was accompanied in males by increased blood urea. Males from the intermediate dose group also showed a slight, but statistically significant increase in this parameter .
Calcium levels were elevated in mid and high dose males, and high dose animals of both sexes showed reduced levels of aspartate aminotransferase, but the biological significance of this change is unclear.

Effects in satellite groups were confined to a slight increase in cholesterol (females) and calcium (males), thus changes in blood chemistry were reversible in this study. No treatment related changes were observed in low dose animals.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of ketones in the urine was noted for high dose animals of both sexes and intermediate dose males . High dose animals also showed increased urine volume and reduced specific gravity . These changes were not apparent in satellite group animals after the treatment-free period .
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weights were markedly increased in the high and mid dose in both sexes. Relative kidney weights were also elevated in high dose animals and mid dose males. in the satellite group, an increase in liver weight was still observed, but kidney weights had returned to normal.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of the high dose group had enlarged livers, which were occasionally dark or showed accentuated lobular pattern, pallor of the adrenals, and abnormally small testes.

Satellite group animals showed patchy pallor of the liver and compacted contents in the caecum and distal ileum. The stomach and small intestine were haemorrhagic with ulceration of the glandular gastric mucosa and white thickening in the nonglandular region of the stomach. Small testes were noted in one animal
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes were observed in the liver, kidneys and testes.

Liver: Periportal hepatocyte vacuolation was observed in two female rats dosed at 750 mg/kg/day. Similar changes were not observed in control animals, nor amongst those from remaining treatment groups, including satellite group animals.

Kidneys: Basophilia and, in some instances, associated dilatation of distal tubules was observed for male and female rats dosed at 750 mg/kg/day. Similar changes were not observed in control animals, nor amongst those from other treatment groups, including satellite group animals.

Testes: Testicular atrophy, frequently bilateral, was seen in all male rats dosed at 750 mg/kg/day. Control animals did not demonstrate similar changes. Although one animal from each of the remaining treatment groups also exhibited a minimal degree of testicular atrophy, this was considered to be spontaneous in origin and unrelated to treatment at these dose levels. Testicular atrophy was also present amongst satellite 750 mg/kg/day animals, although the incidence was reduced (3/5).
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on marked reduction in bodyweight gain, increase in liver (app. +25%) and kidney (app. +13%) weight
Critical effects observed:
not specified

Body and testis weight development:

Dose group/parameter

0 mg/kg

0 mg/kg satellite

50 mg/kg

250  mg/kg

750 mg/kg

750 mg/kg satellite

Body weight

-day 0 [g]

161 ± 8

175 ± 12

168 ± 8

170 ± 8

175 ± 9

161 ± 6

-at end of study [g]

378 ± 15

468 ± 35

372 ± 27

360 ± 30

332 ±13

378 ± 15

Testis weight

Testis weight (absolute) [g]

3.39 ± 0.27

4.64 ± 0.56

3.92 ± 0.30

3.91 ± 0.30

3.09 ± 0.41

3.61 ± 0.87

Testis weight (% body weight)

1.04 ± 0.10

0.97 ± 0.06

1.05 ± 0.08

1.06 ± 0.07

0.91 ± 0.12

0.85 ± 0.23

Incidence and grading of testicular atrophy:

Dose group/ finding

0 mg/kg

0 mg/kg satellite

50 mg/kg

250 mg/kg

750 mg/kg

750 mg/kg satellite

Number of animals

5

5

5

5

5

5

Atrophy testis 1

Grade 1

0

0

4

4

1

0

Grade 2

0

0

1

1

2

2

Grade 3

0

0

0

0

1

0

Grade 4

0

0

0

0

1

0

Grade 5

0

0

0

0

0

1

Atrophy testis 2

Grade 1

0

0

1

0

0

1

Grade 2

0

0

0

0

2

1

Grade 4

0

0

0

0

1

0

Grade 5

0

0

0

0

0

1

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
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

All studies presented have already been discussed in an Annex XV EU dossier for the harmonized classification and labelling of chemical substances, which was submitted by the Federal Institute for Occupational Safety and Health (BauA) in March, 2010 and agreed on by the RAC in October 2010 after memberstate consultation.

In the first 28-day subacute study, 5 rats per sex and group were treated with 50, 250, or 750mg/kg Diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide. The study revealed adverse effects on body weight development, blood, liver and kidney. Body weight gain was reduced by 12% and 15% in the males and females of the mid dose group, respectively, and liver and kidney weights were increased. Clinical signs worsened in the high dose group, were blood chemistry parameters and urinanalysis indicated renal and cholestatic hepatic damage, which was also confirmed by histopathology. Liver and kidney weights were also increased, and body weight gain in this group was decreased by 28% and 19% in males and females. In addition, decreased testes size and testicular atrophy were observed in high dose males. A slight increase in lymphocytes in females hints to inflammatory processes. Though data were within historical control values in this study, inflammation was observed in females in the 90 -day study. No effects were observed in animals treated with 50mg/kg. All changes, except liver weight increase and testes effects in the high dose group, were reversible as demonstrated by a satellite group, that was observed for 14 days after cessation of treatment.

A 90 -day study intended to look for neuropathological effects of the test substance confirmes the results from the 28 -day study. 10 rats per group and sex were treated with dosages comparable to the 28 -day study: 100, 300, or 1000mg/kg of test substance. Again, no effects were observed at the lowest dose. Body weight gain was reduced by 16% in males in the mid dose, while increased liver weights, inflammation and slight anemia were detected in females of this group. 2 females of the high dose died. In this group, body weight gain was reduced by 38% and 16% in male and females, and clinical chemistry indicated liver dysfunction. Testes atrophy was observed with increasing severity in mid and high dose males, whereas neither neurotoxic signs nor functional defects of the animals were reported. The temporary unavailability of the report of the initial 28-day study prompted an investigation of these effects in a second 28-day study with a new batch of the test substance. In contrast to the first test, no adverse effects were observed on the body and testis weight of rats treated with 1000 mg/kg bw/day of Diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide. Histopathology, too, did not reveal any lesions. The 90 -day study, that was conducted with the same dose and batch of test substance, however, did reveal a reduction in size of the testes and epididymes, which was substantiated by the histopathologic lesions found.

Based on the results of one 28-day and the 90-day studies there is clear evidence for lesions of the testes as the only indication that Diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide may lead to reduced fertility of the animals. Due to an administration of the test substance by oral gavage, it cannot be excluded that this may have been a bolus effect. To investigate the toxicokinetics of the substance by a more continuous exposure of the animals, the design of the study was changed to administration of the test substance via the feed. However, the rats avoided the substance-treated feed for palatability reasons, which led to excessive weight loss of the animals, such that they had to be sacrificed on humane grounds. An additional encapsulation to improve acceptance of the test substance did not solve the palatability problem.


Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

The proposed classification for Diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide was already discussed in an EU dossier for the harmonized classification and labelling of substances according to Annex XV, submitted by Germany and received by RAC in March, 2010. Though effects on the testes was confirmed by several studies, they occured only at doses, at which also effects on body weight gain (exceeding a 10% difference) and first signs for liver and kidney dysfunction and thus probably impaired metabolism were observed. In addition, the route of exposure used in the animal test system (gavage) is considered less relevant for human exposure, and bolus effects cannot be excluded. But as mentioned above, a feeding study was impossible to perform, and uptake of the test substance via other routes is unlikely. The proposed classification for effects on fertility of Repro cat. 3 (R62, EU legislation) and Repro cat. 2 (H361f, GHS legislation) was agreed on by the RAC after memberstate consultation in October 2010 and the substance is legally classified accordingly. RAC considered the alternative possibility of classification as Repr 1B (under CLP). However, it was concluded that the effects observed were not severe enough to warrant this classification, taking into account the lack of a consistent effect on the testes across all four repeat dose studies.