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Diss Factsheets

Administrative data

Description of key information

Oral repeated dose toxicity

 

Rats

Key, M-030427-03-1, OECD 407, rat, 21 d,

NOAEL: 100 ppm (corresponding to 9.0 mg/kg bw/day for males and 12.3 mg/kg bw/day for females)

LOAEL: 400 ppm (corresponding to 36.9 mg/kg bw/day for males and 44.6 mg/kg bw/day for females)

 

Key, M-000863-01-1, OECD 408, rat, 13 weeks,

NOAEL: 100 ppm (corresponding to 7.3 mg/kg bw/day for males and and 7.6 mg/kg bw/day females)

LOAEL: 400 ppm (corresponding to 28.6 mg/kg bw/day for males and 35.6 mg/kg bw/day for females)

 

Key, M-003817-02-1, OECD 453, rat, 2 years,

NOAEL: 25 ppm (males, corresponding to 1.2 mg/kg bw/day) and 50 ppm (females, corresponding to 3.3 mg/kg bw/day)

LOAEL: 50 ppm (males, corresponding to 2.5 mg/kg bw/day) and 500 ppm (females, corresponding to 33.5 mg/kg bw/day)

 

Mice

WoE, M-000821-01-1, OECD 407, mice, 14d,

NOAEL: 200 ppm (corresponding to 84.3 mg/kg bw/day for males and 113.1 mg/kg bw/days for females)

LOAEL: 2000 ppm (corresponding to 765.1 mg/kg bw/day for males and 1201.2 mg/kg bw/days for females)

 

WoE, M-000688-01-1, OECD 407, mice, 21d,

NOAEL: 100 ppm (corresponding to 30.1 mg/kg bw/day for males and 63.9 mg/kg bw/days for females)

LOAEL: 1000 ppm (corresponding to 367.8 mg/kg bw/day for males and 559.3 mg/kg bw/days for females)

 

Key, M-000697-02-1, OECD 408, mice, 14 weeks,

NOAEL: 50 ppm (males, corresponding to 19.9 mg/kg bw/day), no NOAEL established for females,

LOAEL: 250 ppm (males, corresponding to 102.6 mg/kg bw/day) and 50 ppm (females, corresponding to 27.2 mg/kg bw/day)

 

Dogs

Key, M-003814-01-1, OECD 409, dogs, 13 weeks,

NOAEL: 250 ppm (males, corresponding to 8.5 mg/kg bw/day), 2000 ppm (females, corresponding to 65.3 ppm)

LOAEL: 1000 ppm (males, corresponding to 34.9 mg/kg bw/day)

 

Key, M-003818-01-1, OECD 452, dogs, 52 weeks,

NOAEL: 250 ppm (males, corresponding to 8.88 mg/kg bw/day), 1000 ppm (females, corresponding to 33.8 mg/kg bw/day)

LOAEL: 1000 ppm (males, corresponding to 34.42 mg/kg bw/day)

 

Inhalation repeated dose toxicity

Key, M-241815-01-1, similar to OECD 412, rat, 28 d,

NOAEC (systemic): 18.2 mg/m³ (dust)

LOAEC (systemic): 143.2 mg/m³ (dust)

NOAEC (respiratory system, local): 143.2 mg/m³ (dust)

 

Dermal repeated dose toxicity

Key, M-000824-01-1, OECD 410, rat, 28 d,

NOAEL (systemic, males): 100 mg/kg bw/day, LOAEL (systemic, males): 300 mg/kg bw/day

NOAEL (systemic, females): 300 mg/kg bw/day, LOAEL (systemic, females): 1000 mg/kg bw/day

NOAEL (dermal, local, males, females): 1000 mg/kg bw/day

Key value for chemical safety assessment

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:
19 Jan - 14 Feb 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2008
Deviations:
yes
Remarks:
The study was conducted to investigate the mechanism of thyroidal changes after treatment with the test material. Therefore, the examinations were limited. For details please refer to "Principles of method if other than guideline".
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1995
Deviations:
yes
Remarks:
The focus was set on the mechanism of test substance-related thyroidal changes.
Principles of method if other than guideline:
The study period was 3 weeks instead of 4, animals were housed individually instead of in groups, and were checked for morbidity and moribundity only once per day. Sensory reactivity was not assessed and no haematology was performed. Regarding clinical chemistry, only T3, T4, TSH, UDP-GT-levels and activity and plasma hormone levels were assessed. Organ weights were recorded for liver, uterus and the left thyroidal lobe only and histopathology was performed only for thyroid.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Common strain for toxicological studies and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: mean of male groups: 173-178 g, mean of female groups: 131-135 g
- Fasting period before study: not applicable
- Housing: individually under conventional conditions in type IIa polycarbonate cages on low-dust wood granulate (S sniff Spezialdiäten GmbH, Soest, Germany), during acclimatization rats were kept in groups (about 5 animals per type III cage, separated by sex)
- Diet: fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly checked for contaminations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 55
- Air changes (per hr): 15.20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany)
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: peanut oil was mixed in the feed to prevent dust-formation
- Amount of vehicle in feed: 1%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on homogeneity and stability of the test substance in the administration vehicle covering the concentration range used were obtained. It was found that the test substance was stable in the diet for 8 days.
Duration of treatment / exposure:
21 days
Frequency of treatment:
continuously via the diet
Dose / conc.:
25 ppm
Remarks:
corresponding to 2.6 and 3.1 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
100 ppm
Remarks:
corresponding to 9.0 and 12.3 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
400 ppm
Remarks:
corresponding to 36.9 and 44.6 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
1 600 ppm
Remarks:
corresponding to 145.1 and 190.8 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The same doses had been administered in a subchronic feeding study in rats. In the present study, particular emphasis was given to the effects in thyroids and on thyroid hormones.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: Body surfaces and orifices, posture, general behavior, breathing and excretory products

BODY WEIGHT: Yes
- Time schedule for examinations: Before beginning of the study and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days -2 through 14 (retroorbital plexus) and Day 21 and 22 (cardiac puncture)
- Anaesthetic used for blood collection: Yes (deep diethylether anesthesia)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Triiodothyronine (T3), thyroxine (total) (T4), thyroxine binding capacity (TBC) and thyroid stimulating hormone (TSH) levels and protein content (from heparinized plasma), UDP-glucuronyl transferase (UDP-GT, from liver samples at time of necropsy)

PLASMA/SERUM HORMONES/LIPIDS: Yes, see above

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
Sacrifice of surviving animals was performed by exsanguination under deep ether anesthesia,

GROSS PATHOLOGY: Yes
Organ weights were recorded for liver, uterus and the left thyroidal lobe.


HISTOPATHOLOGY: Yes
tissues collected: liver, thyroid, uterus and pinnae were fixed in aqueous 10% formalin solution, tissues investigated: thyroid
Optional endpoint(s):
Optional endpoints: No
Other examinations:
None
Statistics:
The quantitative results for individual animals were used to calculate group means, median and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by '+' for p < 0.05 and '++' for p < 0.01.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only effect observed was bright feces of one female (high dose) after 2 and 3 weeks. This finding was considered incidental.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: decreased transient body weight (males, Week 1 and 2, -10.5 and -8.4%) and decreased body weight in females (throughout the study, -12.5 to -13.0%), reduced body weight gains in males (-14.9%) and females (-43.5%), all compared to controls.

Summarized data can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: reduced feed consumption and daily feed intake compared to controls during Week 1 for both sexes

Not treatment related:
Decreased daily feed intake for the 100 and 400 ppm male rats compared to controls was not considered biologically relevant because of a lack of dose response.

Summarized data for the 21-day interval can be found in Attachment 2 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid parameters
- 1600 ppm: significant reduction in T3 levels on Day 2 in male and female rats, significant reduction in total T4 levels on Days 2 and 7 in male rats and on Day 2 in female rats, increase in the level of TSH in males on Day 14 and in females on Days 7 and 22, all compared to controls.

As can be expected, there was variation within the observations made in the control groups at different times. Hormone levels underlie a wide intra-individual variation. For relative changes, the pre-treatment data of each treatment group was used instead of the control group as reference. Therefore, analysis of covariance has been made. Treatment-related results were

- 400 ppm: increased thyroid stimulating hormone (TSH) in males from Day 7-21 (not statistically significant but in dose-dependency with results seen at 1600 ppm) compared to controls.
- 1600 ppm: reductions in T3 and T4 on Day 2 for males (-21.2 and -32.0%, respectively) and females (-30.9 and -34.4%, respectively) compared to controls, reduction of T4 continued for males throughout the study (-11.31 to -38.88%) and for females at Day 7 and 22 (-21.8 and -41.6%, respectively) compared to controls, increased TSH throughout the study in males and females, statistically significant at Day 14 in males (532.8%) and Day 7 (127%) and Day 22 (247.7%) in females compared to controls.

Summarized data can be found in Attachment 3.

UDP levels
- 400 ppm: increased UDP-GT levels in males (+98.2%) and females (+64.1%) compared to controls.
- 1600 ppm: increased UDP-GT levels in males (+252%%) and females (+221%%) compared to controls.

Summarized data can be found in Attachment 4. Reference values for total protein and thyroid parameters are presented in Attachment 5.

Further findings not considered to be treatment-related:
Changes in plasma protein levels were statistically significant compared to controls at some instances (females at 1600 ppm, males at all dose levels). However, these were not dose-dependent and close to control values and were therefore not considered treatment-related.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 400 ppm: increased absolute liver weight in males compared to controls (+14.2%)
-1600 ppm: increased absolute (+32.2% in males, +22.9% in females) and relative (+40.6% in males, +39.4% in females) liver weight compared to controls

Further findings not considered to be treatment-related:
The decreased thyroid weights in females (absolute at > 400 ppm, relative at 400 ppm) compared to controls were not regarded as treatment-related, as there was no dose response for the relative thyroid weight.

Summarized data can be found in Attachment 6.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: increased liver (10/10 males, 9/10 females), marked lobular pattern (7/10 females)

Summarized data is presented in Attachment 7.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 400 ppm: minimal to slight hypertrophy of the follicular epithelium compared to controls (males)
- 1600 ppm: minimal to slight hypertrophy of the follicular epithelium compared to controls (males/females)

Summarized data can be found in Attachment 8 (attached background material).
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
other: corresponding to 9.0 mg/kg bw/day for males and 12.3 mg/kg bw/day for females
Key result
Dose descriptor:
LOAEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 36.9 mg/kg bw/day for males and 44.6 mg/kg bw/day for females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The present study was conducted according to the OECD guideline 407 (dated 1995), however with the focus being set on the mechanism of thyroidal changes related to the test substance; the study conduct was GLP compliant. It was shown that the test substance induces the hepatic enzyme UDP-glucuronyl transferase (UDP-GT). In general, induction of UDP-GT leads to increased T4 glucuronide formation and excretion of T4, resulting in decreased T3 and T4 levels, which was also found in this study. TSH levels increased to compensate for decreased T4 and T3 levels and led to a continuous activation of the thyroid as already seen by hypertrophy of the follicular epithelium of the thyroid. Moreover, changes in the liver and on body weight and feed consumption were observed. It was concluded that the activation of UDP-GT was seen as a primary effect of the test substance and the consequent changes in hormone level and thyroid histopathology secondary effects due to activation of the hypothalamic-pituitary-thyroidal (HPT) axis. A dietary concentration of 100 ppm (equivalent to 9.0 and 12.3 mg/kg body weight per day, for males and females, respectively) was determined as a clear no-observed-effect level.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 Mar - 12 Apr 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2008
Deviations:
yes
Remarks:
Treatment duration was 21 days, only 3 animals/sex, some recommended parameters not considered (hematology, clinical chemistry, ophthalmology), histopathology not assessed, organ weights only determined for liver and kidney
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Commonly used for toxicology studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Bomholtgaard Breeding Research Center Ltd, Ry, Denmark
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 19-23 g (males), 16-18 g (females)
- Fasting period before study: no
- Housing: during the study period, animals were kept individually in Makrolon® type I cages on low-dust wooden granulate (Ssniff, Soest/Westphalia, Germany), during acclimation animals were kept in groups in Makrolon® type III cages
- Diet: "fixed-formula" standard diet (Altromin® 1321, from Altromin GmbH and
Co KG, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were analyzed regularly, no contaminations were found

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Mar To: 12 Apr 1994
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): twice, rations were changed every week
- Mixing appropriate amounts with granulated feed: Altromin® 1321, peanut oil (1%) was added to prevent dust formation
- Storage temperature of food: room temperature during feeding period, otherwise frozen

VEHICLE
peanut oil, 1% in diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogenity of the test substance in the feed were analyzed before and during the study and was confirmed for the period of use.
Duration of treatment / exposure:
21 days
Frequency of treatment:
continuously via the diet
Dose / conc.:
100 ppm
Remarks:
corresponding to 30.1 and 63.9 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 367.8 and 559.3 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
10 000 ppm
Remarks:
corresponding to 4141.0 and 5785.1 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: This study was intended as dose range-finder for a subsequent subchronic study. Thus, doses from 100 to 10000 ppm were selected to allow for the detection of possible effects and a dose-response relationship.

Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and public holidays)
- Parameters checked: body surfaces, orifices, posture, general behavior, breathing and excretions

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
All surviving animals were sacrificed after 21 days by exsanguination in deep diethyl ether anesthesia.

GROSS PATHOLOGY: Yes
Organ weights were recorded for liver and kidney

HISTOPATHOLOGY: Tissues were collected but analysis was not performed
- fixed in: 10% buffered formaldehyde, lung and urinary bladder were instilled with 10% buffered formaldehyde solution
- tissues sampled: adrenal glands, aorta, bone marrow (in femur and sternum), brain (in toto), caecum, colon, duodenum, epididymides, extraorbital lacrimal gland, eyelids, eyes, femur with knee joint, gall bladder, Harderian glands, head, heart, ileum, jejunum, kidneys, larynx, liver, lung, lymph nodes (mandibular, mesenteric), mammary glands (with skin), oesophagus, optic nerve, ovaries, oviducts, pancreas, pituitary, prostate, rectum, residual intestine, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle (femoral), skin, spinal cord (vertebral column; cervical, thoracic, lumbar), spleen, sternum, stomach, tattooed auricles, testes, thymus (if present), thyroid + parathyroid, tongue, trachea, ureter, urethra, urinary bladder, uterus, vagina, Zymbal's gland
Optional endpoint(s):
None
Other examinations:
None
Statistics:
The arithmetic group means and standard deviations were calculated from individual animal data for the body weights, food and water intake as well as for organ weights.

Since groups consisted of only 3 individuals no statistical evaluation of results was performed.
Clinical signs:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment animals.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 10000 ppm: mean body weight was reduced in males compared to controls (-4.3 to -8.3%)

Summarized results can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: decreased compared to controls in females (-9%)
- 10000 ppm: increased feed intake in males compared to controls (+14%), decreased compared to controls in females (-10%)

Summarized data can be found in Attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 10000 ppm: decreased compared to controls in females (-17%)

Summarized results can be found in Attachment 3.
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: increased absolute (10% males, 9% females), and relative (18% males, 9% females) liver weights, compared to controls.
- 10000 ppm: increased absolute (45% males, 25% females), and relative (59% males, 31% females) liver weights, compared to controls

Regarding the kidney, no treatment-related effects occurred. Relative kidney weights in females was slightly lower (5%) compared to controls but this finding is regarded as incidental due to the low difference and the low number of animals tested.

Summarized results can be found in Attachment 4.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 10000 ppm: enlarged liver (2/3 males)
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 30.1 mg/kg bw/day for males and 63.9 mg/kg bw/days for females.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
gross pathology
organ weights and organ / body weight ratios
water consumption and compound intake
Remarks on result:
other: corresponding to 367.8 mg/kg bw/day for males and 559.3 mg/kg bw/days for females.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
A 21 day feeding study was conducted to assess the toxicity of the test material to the mouse after repeated administration. The study was performed under GLP and was intended as a dose range-finder experiment for a subsequent subchronic toxicity study in mice. The study duration was 21 days and the nominal test concentrations were set at 0, 100, 1000 and 10000 ppm in diet. It was found that the test material exhibited effects at 1000 ppm and above in male and female mice, as seen by altered body weight, feed and water consumption as well as altered liver weight and appearance. The effects on the liver are probably linked to hepatic enzyme induction. Therefore, the NOAEL was set at 100 ppm (corresponding to 30.1 mg/kg bw/day for males and 63.9 mg/kg bw/day for females).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Apr - 5 Aug 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Missing examinations, for details please refer to "Principles of method if other than guideline"
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
Animals were housed individually instead of in groups, feed was not analyzed for phytoestrogens, no ophthalmological examinations and no sensory reactivity were assessed, animals were not fasted prior to sacrifice, thyroid parameters were not examined (T3, T4, TSH, thyroid weight), estrus cycle was not determined, prostate, uterus, thymus, spleen and pituitary weight were not investigated.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Commonly used for toxicological studies and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Bomholtgaard Breeding Research Center Ltd, Ry, Denmark
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22 g (males, mean), 18 g (females, mean)
- Fasting period before study: not applicable
- Housing: animals were kept individually in Makrolon® type I cages on low dust wooden granulate (Ssniff, Soest/Westphalia, Germany)
- Diet: "fixed-formula" standard diet (Altromin® 1321, from Altromin GmbH and Co KG, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were analyzed regularly, no contaminations were found

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with granulated feed: Altromin® 1321, peanut oil (1%) was added to prevent dust formation
- Storage temperature of food: room temperature during feeding period, otherwise frozen

VEHICLE
peanut oil, 1% in diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test substance in the feed were analyzed using High Performance Liquid Chromatography (HPLC). Analysis was performed on the day of mixing and after 7 days at room temperature. Homogeneity and stability were confirmed for the duration of the study.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
continuously via the diet
Dose / conc.:
50 ppm
Remarks:
corresponding to 19.9 and 27.2 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
250 ppm
Remarks:
corresponding to 102.6 and 139.1 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
1 250 ppm
Remarks:
corresponding to 542.4 and 704.3 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
6 250 ppm
Remarks:
corresponding to 2819.9 and 3351.0 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were based on the subacute range finding study (M-000688-01-1) conducted with the same mouse strain treated orally with 100, 1000 and 10000 ppm of the test substance in the feed over 21 days. In this study, 100 ppm was the NOAEL, above that, alterations in body weight, feed and water consumption and liver weight and appearance were observed. Therefore, the doses in the present study were set as 50, 250, 1250 and 6250 ppm.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and public holidays)
- Parameters checked: body surfaces, orifices, posture, general behavior, breathing and excretions

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 12, from one of the caudal veins (glucose) or retro-orbital venous plexus (all other parameters)
- Anesthetic used for blood collection: Yes (ether, for blood withdrawn for all parameters except glucose) / No (for blood withdrawn for glucose analysis)
- Animals fasted: No
- How many animals: all
- Parameters checked: differential blood count, erythrocyte cell count, hemoglobin concentration, hematocrit, white cell count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean cell volume (MCV), platelet count, thromboplastin time (hepato Quick-test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13, liver samples were taken at necropsy
- Animals fasted: No
- How many animals: all
- Parameters checked: Alkaline phosphatase (APh), alanine and aspartate aminotransferase (ALAT and ASAT), glucose, albumin, bilirubin, cholesterol, creatinine, total protein, triglycerides, urea, N-Demethylase, cytochrome P450

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights were recorded for adrenals (in pairs), heart, testes (in pairs), brain, liver, spleen, kidneys (in pairs), ovaries (in pairs)

HISTOPATHOLOGY: Yes
- fixed in: 10% buffered formaldehyde, lung and urinary bladder were instilled with 5% buffered formaldehyde solution
- tissues collected: aorta, adrenals, bone marrow (in femur and sternum), brain (in toto), caecum, colon, duodenum, epididymides, extraorbital glands, eyelids, eyes, femur with knee joint, gall bladder, Harderian glands, head, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (mesenteric, mandibular), mammary glands, musculature (femoral), esophagus, optic nerve, ovaries, oviducts, pancreas, pituitary, prostate, rectum, residual intestine, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord (vertebral column: cervical, thoracic, lumbar), spleen, sternum, stomach, tattooed auricles, testes, thymus (if present), thyroid & parathyroids, tongue, trachea, ureter, urethra, urinary bladder, uterus, vagina, Zymbal glands
- animals examined: control and high dose group (for liver and kidney: all animals of all dose groups)
- tissues examined: liver, kidney, pituitary and thyroid glands, epididymides, testes as well as all organs and tissues
with gross pathological findings
- embedding media: paraplast
- thickness of sections: 5 µm
- staining: hematoxylin and eosin
Optional endpoint(s):
None
Other examinations:
None
Statistics:
The arithmetic group means and standard deviations were calculated from individual animal data.
Data of treated groups were compared to those of the corresponding control group by means of the significance test (U-test) and the Mann-Whitney-Test (significance level of 5% and 1% (two tailed). Significant differences to controls are indicated with */ + for p < 0.05 and **/++ for p < 0.01.
Clinical signs:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups were found.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 female of the control group died, 1 male of the 1250 ppm group was killed in moribund state, 7 animals died most probably due to blood sampling (2 control males, 1 male of the 250 ppm group, 2 males of the 6250 ppm group, 1 female of the 50 ppm group, 1 female of the 250 ppm group).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 50, 250 and 1250 ppm: No treatment related effects were obserded compared to the control group up to and including 1250 ppm (both sexes).
- 6250 ppm: statistically significantly reduced body weight in males compared to controls (up to -14%), females were not affected up to and including the highest dose level.

Summarized results can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 50 and 250 ppm: No treatment related effects were obserded compared to the control group up to and including 250 ppm (both sexes).
- 1250 ppm: increased feed consumption in males compared to controls (+12%), reaching statistical significance in Week 3, and Week 8 - 11.
- 6250 ppm: increased feed consumption in males compared to controls (+8%), reaching statistical significance in Week 2 - 4, 8, 10 and 12.

Females were not affected up to and including the highest dose level.

Summarized results can be found in Attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 50, 250 and 1250 ppm: No treatment related effects were obserded compared to the control group up to and including 1250 ppm (both sexes).
- 6250 ppm: individual water intake was statistically significantly decreased up to 30% in males (all weeks of the study), and up to 12% in females (Week 11, reaching statistical significance in Weeks 2, 5, 7, 8, 11, 13 and 14) compared to controls.

Summarized results can be found in Attachment 3.
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 50 and 250 ppm: No treatment related effects were obserded compared to the control group up to and including 250 ppm (both sexes).
- 1250 ppm: marginally, but statistically significantly decreased hematocrit values compared to controls (males), no effects were observed in females.
- 6250 ppm: marginally, but statistically significantly decreased hematocrit values compared to controls (males. females)

Other findings like altered Hepato-Quick Test in males at 50 and 250 ppm or decreased MCV in males and females at 250 ppm and above were not considered toxicologically relevant because they were not dose dependent and/or based on individual values within the 2s-range of the respective parameter. Markedly less nuclei shadows in differential blood count compared to controls in the high dose group (males) was also considered incidental because increased lymphocyte counts are in general not critical.

Summarized data can be found in Attachment 4.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: No treatment related effects were obserded compared to the control group at 50 ppm (both sexes).
- 250 ppm: statistically significantly increased N-demethylase activity (males), compared to controls. In females, no effects were observed.
- 1250 ppm: statistically significantly reduced bilirubin concentration (males, females), statistically significantly increased N-demethylase activity (males, females), statistically significantly increased cytochrome P450 content (males, females), all compared to controls
- 6250 ppm: statistically significantly decreased cholesterol (males, females), statistically significantly increased triglycerides (males), statistically significantly reduced bilirubin concentration (males, females), slightly but statistically significantly reduced protein and albumin concentrations (females), statistically significantly increased N-demethylase activity (males, females), statistically significantly increased cytochrome P450 content (males, females), all compared to controls

Not treatment related or toxicologically relevant:
At 250 and 1250 ppm, females had lower cholesterol levels but this was without relevance since all individual values are within the 2s range of reference values of the laboratory (Attachment 7). At 50, 250 and 1250 ppm, females had reduced plasma protein levels but all (but one) individual values were within the normal range and control females showed relatively high protein concentrations.

Summarized results can be found in Attachment 5.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 50 and 250 ppm: No treatment related effects were obserded compared to the control group up to and including 250 ppm (both sexes).
- 1250 ppm: statistically significantly increased absolute (+8% for females) and relative (+9% for males, +10% for females) liver weights compared to controls
- 6250 ppm: statistically significantly increased absolute (+19% for males, +40% for females) and relative (+39% for males, +42% for females) liver weights, all compared to controls

Other differences that were statistically significant different from controls but not dose dependent or due to weight differences and therefore not considered toxicologically relevant were decreased absolute brain weight in 1250 ppm males, reduced relative brain weight at 6250 in males and lower mean absolute kidney weights in males at 250 ppm and above. The increased relative heart weight (+12% females at 6250 ppm) had no histopathological correlation so that it was considered incidental. The adrenal weights of females were slightly (but statistically not
significantly) increased at ≥ 1250 ppm.

Summarized results can be found in Attachment 6.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 50 ppm: enlargement of the adrenal X zone (females), compared to controls
- 250 ppm: hepatocellular hypertrophy (males), enlargement of the adrenal X zone (females), all compared to controls
- 1250 ppm: hepatocellular hypertrophy (males, females), reduction or lack of vacuoles in epithelial cells of the proximal tubule (males), enlargement of the adrenal X zone (females), all compared to controls
- 6250 ppm: hepatocellular hypertrophy (males, females), reduction or lack of vacuoles in epithelial cells of the proximal tubule (males), enlargement of the adrenal X zone (females), all compared to controls
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on results:
Historical control data can be found in Attachment 7. Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 8 and 9).
Key result
Dose descriptor:
NOAEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: corresponding to 19.9 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
< 50 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Effects were seen at the lowest dose level.
Remarks on result:
other: corresponding to < 27.2 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
water consumption and compound intake
Remarks on result:
other: corresponding to 27.2 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
water consumption and compound intake
Remarks on result:
other: corresponding to 102.6 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The present study was intended as a preliminary dose range-finding study for the subsequent 2-year carcinogenicity study to be conducted with mice. This subchronic study with a duration of 14 weeks was conducted according to the OECD guideline 408 (dated 1981) and GLP conditions. The test item was offered to male and female mice at 50, 250, 1250 and 6250 ppm in diet.
For the females, vacuolisation, hypertrophy and enlargement of the adrenal X-zone were decisive for setting the LOAEL at the lowest tested concentration of 50 ppm, equivalent to 27.2 mg/kg bw/day; for the males, the LOAEL was set at 250 ppm equivalent to 102.6 mg/kg bw/day, based on liver enzyme induction and hepatocellular hypertrophy.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 - 27 Jul 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2008
Deviations:
yes
Remarks:
Study duration shorter than recommended, no hematology analysis was performed, clinical chemistry was limited to liver specific markers, organ weights were only determined for the liver and histopathology was performed for the liver, only.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
Commonly used for toxicology studies, recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Bomholtgaard Breeding and Research Centre Ltd
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 18-23 g (males), 17-19 g (females)
- Fasting period before study: no
- Housing: individually in Polycarbonate cages Type I with low-dust wood shavings type S 8/15 (supplier: Ssniff, Spezialdiaten GmbH, Soest/Westphalia, Germany), in groups of 5 during acclimation in Polycarbonate cages Type III
- Diet: fixed-formula standard diet ("Altromin® 1321 Diet for Rats and Mice", Altromin GmbH and Co. KG, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Feed and water were routinely checked for contaminations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 40-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with granulated feed: test substance was blended into the diet with a pelletizing mixer, peanut oil was mixed in (1% concentration) to prevent dust formation.
- Storage temperature of food: room temperature

VEHICLE
Peanut oil, 1% in diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the feed was verified for two different dates. Homogeneity and stability were analyzed and confirmed. The concentration in feed was determined to be 48.6, 204, 1930 and 9220 ppm.
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily/7 days per week
Dose / conc.:
50 ppm
Remarks:
corresponding to 21.6 and 29.8 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
200 ppm
Remarks:
corresponding to 84.3 and 113.1 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
2 000 ppm
Remarks:
corresponding to 765.1 and 1201.2 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
10 000 ppm
Remarks:
corresponding to 4143.2 and 5449.8 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected based on the data obtained in a previous subacute rat study.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS AND CLINICAL OSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and holidays)
- Cage side observations: Body surfaces and orifices, posture, general behavior, breathing and excretory products were assessed

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the time of necropsy from the retroorbital venous plexus
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: alanine aminotransferase (ALAT), alkaline phosphatase (APh), aspartate aminotransferase (ASAT), glutamate dehydrogenase (GLDH), gamma-glutamyltransferase (GGT), albumin (ALB), bilirubin (BILI-t), cholesterol (CHOL), total protein (PROT), Cytochrome P-450 monooxygenases: 7-ethoxycoumarin-deethylase (ECOD), 7-ethoxyresorufin-deethylase (EROD), aldrin-epoxidase (ALD); Phase Il-enzymes: epoxide hydrolase (EH), glutathione S-transferase (GST), UDP-Glucuronyl-transferase (UDP-GT)

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
All surviving animals were sacrificed by exsanguination under deep ether anesthesia.

GROSS PATHOLOGY: Yes
Organs weighed: livers

HISTOPATHOLOGY: Yes

Liver and physical identifier (tattooed ears) were fixed in aqueous Bouin solution.
Additionally, one liver lobe of all mice were fixed in 10 % aqueous formaldehyde solution.
Optional endpoint(s):
None
Other examinations:
None
Statistics:
Arithmetic group means and standard deviations were calculated for all groups. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p<0.05 and "++" for p<0.01. In case of numbers of values too low to calculate test statistics this is indicated by "-".

Parametric measurements were compared using ANOVA followed by a Dunnett's test. These were body weight, feed intake, water consumption and organ weight data (relative organ weights subsequent to logarithmic transformation).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only finding was in one male of the 2000 ppm group that got caught within the automatic feeder
inside the cage and strangulated itself, decreased motility and reactivity, and labored breathing were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The only animal that died was the one described above.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences were found between control and treatment groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences were found between control and treatment groups.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 2000 ppm: decreased water consumption in males compared to controls
- 10000 ppm: decreased water consumption in males compared to controls

Summarized results can be found in Attachment 1.
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 200 ppm: increased ECOD, ALD, GST, UDP-GT in females compared to controls
- 2000 ppm: increased ECOD, EROD, ALD, GST, UDP-GT in males and females compared to controls
- 10000 ppm: increased ECOD, EROD, EH, ALD, GST, UDP-GT in males and females compared to controls

Not treatment related:
At 10000 ppm, decreased cholesterol (males), increased concentration of protein (males), decreased concentration of bilirubin and albumin (females) were observed, all compared to controls. These changes were statistically significant to the controls but occurred in one sex only, and/or all individual values were within the 2s range, and/or the differences to the controls were very minor.

Summarized data for liver samples can be found in Attachment 2.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 2000 ppm: increase in absolute and relative liver weights compared to control for males (+14.6% absolute, +18.4% relative) and females (+17.5% absolute, +21.7% relative)
- 10000 ppm: increase in absolute and relative liver weights compared to control for males (+15.9% absolute, +24.9% relative) and females (+32.6% absolute, +38.7% relative)

Summarized results can be found in Attachment 3.
Gross pathological findings:
no effects observed
Description (incidence and severity):
not applicable
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 200 ppm: hypertrophy of centrolobular hepatocytes with more amorphous cytoplasm of the affected liver cells (males)
- 2000 ppm: hypertrophy of centrolobular hepatocytes with more amorphous cytoplasm of the affected liver cell (males, females), slightly increased minute vacuolar fat storage (males, females)
- 10000 ppm: hypertrophy of centrolobular hepatocytes with more amorphous cytoplasm of the affected liver cell (males, females), slightly increased minute vacuolar fat storage (males, females)
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 84.3 mg/kg bw/day for males and 113.1 mg/kg bw/days for females
Key result
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 765.1 and 1201.2 mg/kg bw/day actual dose ingested for males and females, respectively
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
To assess the effects of the test substance, a subacute oral toxicity study was conducted in mice according to OECD 407 (dated 1981) and under GLP. The study duration was 14 days and the nominal test concentrations were set at 0, 50, 200, 2000 and 10000 ppm in diet. Under the conditions of the test, the test substance had no influence on body weight, feed consumption and mortality. Water intake was reduced at 2000 ppm an above in males. Hepatic effects of the test substance were evident as dose-dependent increase of phase I and phase II enzymes, starting from 2000 ppm in males and females. Probably secondary effects to this induction were increased liver weight in males and females at 2000 ppm and above and histopathological alteration of hepatocytes (starting at 2000 ppm in females). THE NOAEL was set at 200 ppm (corresponding to 84.3 mg/kg bw/day for males and 113.1 mg/kg bw/days for females).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Jan - 9 Mai 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted 1998
Deviations:
yes
Remarks:
Liver was weighed without gall bladder, ornithite decarboxylase was not determined. These deviations however are considered minor.
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 19 - 20 weeks
- Weight at study initiation: 6.1 - 8.9 kg
- Fasting period before study: not applicable
- Housing: individually, all animals were allowed to exercise for 60 min daily, except at weekends and public holidays.
- Diet: "Ssniff HH sole diet for dog husbandry, double ground" (Ssniff Versuchstierdiäten GmbH, Soest, Germany), 300 g per day
- Water: tap water, ad libitum
- Acclimation period: 18 days

DETAILS OF FOOD AND WATER QUALITY:
The nutritive composition and contaminant content of the standard diet were routinely spot-checked and analyzed. The tests were inconspicuous. Drinking water was also analyzed and the analysis yielded no evidence for an altered quality of the tap water used.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 30 – 60
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Jan To: 8 Mai 1995
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly, daily mixed with water to form the feed paste (see below)
- Mixing appropriate amounts with standard diet; the dry feed was mixed with the test substance at the respective concentrations. Immediately before administration, hand-warm tap water was added to the feed meal (control) and the feed-substance mixtures at a ratio of 1:1 to obtain a homogenous paste. This was given to the animals daily in the morning. Remaining feed that was not consumed after 20 to 22 hours was weighed.
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance in the feed was tested before study initiation. It was found that the test substance was stable and evenly distributed for 14 days in the dry diet and 24 hours in the moist feed.
For analytical monitoring, the active ingredient (range of concentrations: 10 mg a.i./kg to 2500 mg a.i./kg dry feed and 10 mg a.i./kg to 1000 mg a.i./kg dry feed with 50% water) was extracted from the vehicle (either dry feed or feed with 50% water) with 100 mL of methanol applying an empty Extrelut® Column. The concentration of active ingredient was quantitatively determined by high performance liquid chromatography (HPLC) and UV-detection. The recoveries for the active ingredient ranged between 10.3 - 2460 mg a.i./kg food and were in a range of 58.1 to 99.0 %. The relative standard deviation was in a range of 1.3 - 6.2%. Thus, the method was considered valid for the concentration range of about 10 to 2500 mg a.i./kg food.
Duration of treatment / exposure:
Control, low (250 ppm)and mid (1000 ppm) concentration groups: 15 weeks
High concentration group: 13 weeks
Hereby it has to be noticed that the animals of the high concentration group were treated with 4000 ppm during the first 4 days. Due to severe effects (vomiting and body weight reduction) treatment was stopped and the animals were allowed to recover over a period of 10 days, received normal diet not supplemented with the test item. Thereafter the highest concentration level was reduced to 2000 ppm for the remaining duration of the experiment. Therefore, the whole study period was extended for 2 weeks and accordingly, the number of treatment days for all groups was about 105 to 106 days.
Frequency of treatment:
continuously via the diet
Dose / conc.:
250 ppm
Remarks:
corresponding to 8.5 mg/kg bw/day for males and 8.9 mg/kg bw/day for females, combined 8.7 mg/kg bw/day
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 34.9 mg/kg bw/day for males and 34.7 mg/kg bw/day for females, combined 34.8 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
corresponding to 68.0 mg/kg bw/day for males and 65.3 mg/kg bw/day for females, combined 66.7 mg/kg bw/day
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were based on a previously conducted subacute oral toxicity study in dogs in which the test substance was administered via the feed at concentrations of 100, 300 and 1000 ppm to male and female beagle dogs over 4 weeks. The 1000 ppm concentration was increased to 2500 ppm during the study and an additional group received 2500 ppm in their feed throughout 4 weeks. No relevant toxicological findings were observed but a slight decreased feed intake at 1000/2500 ppm. Therefore, the doses for the present study were initially as follows: 250, 1000 and 4000 ppm.
Positive control:
no
Observations and examinations performed and frequency:
GENERAL OBSERVATIONS: Yes
- Time schedule: daily (repeatedly)
- observations: appearance and behavior

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: two weeks before start of treatment and in Weeks 7 and 15
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of the study (Week -3, -2) and in Weeks 2, 7 and 15
- Anesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: differential blood count, erythrocyte morphology (= red blood cell morphology), erythrocyte count (= red blood cell count; ERY), hemoglobin concentration in the blood (HB) , hematocrit (= packed cell volume; HCT), Heinz bodies, leukocyte count (= white blood cell count; LEUCO), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), methemoglobin (MET-HB), reticulocytes (RETI), thrombocyte count (= platelet count; THRO), partial thromboplastin time (PTT), thromboplastin time (HepatoQuick; HQUICK)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above, liver samples were collected at time of necropsy
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALAT), alkaline phosphatase (APh), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), creatine kinase (CK), glutamate dehydrogenase (GLDH), lactat dehydrogenase (LDH), albumin (ALB), bilirubin (BILI-t), cholesterol (CHOL), creatinine (CREA), total protein (PROT), triglycerides (TRIGL), urea (UREA), glucose (GLUCOSE), chloride (Cl), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), magnesium (Mg), iron (Fe), triiodothyronine (T3), thyroxine (T4), thyroxine-binding capacity (TBC), thyroid stimulating hormone (TSH)
- Parameters checked in liver samples: N- and O-Demethylase, triglycerides, Cytochrome P-450 content

URINALYSIS: Yes
- Time schedule for collection of urine: before the start of the study (Week -2) and in Weeks 2, 7 and 15
- Metabolism cages used for collection of urine: Yes (for 6 h)
- Animals fasted: Yes (during collection)
- Parameters checked: creatinine (CREA), density, sodium (Na), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Electrocardiograms (ECG)
- Time schedule: two weeks before start of treatment and in Weeks 7 and 15 (before and 2 h after application)
Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination under Narcoren® anesthesia.

GROSS PATHOLOGY: Yes
At necropsy, the animals were first subjected to gross pathological examination. After that, the following organs were weighed: brain, heart, liver, lungs, spleen, adrenals, kidneys, pancreas, thyroid, pituitary, testes, prostate gland, uterus, thymus and ovaries

HISTOPATHOLOGY: Yes
- fixed tissues: adrenal glands, aorta, epididymides, esophagus, eyes, femur, gall bladder, heart (two papillary muscles of the left ventricle), intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, liver, lung (one lobe was instilled with 4% formaldehyde), lymph nodes (mandibular and mesenteric), optic nerves, ovaries, oviducts, pancreas, pituitary gland, prostate, salivary glands (parotid and mandibular gland), sciatic nerve, skeletal muscle (thigh), skin (mammary area with nipple), spleen, sternum, stomach (fundic and pyloric region), testes, thymus, thyroid gland with parathyroid gland, tongue, tonsils, trachea, urinary bladder, uterus, vagina and all organs/tissues with macroscopic alterations.
- lung, brain, two further liver pieces, a transversal slice of kidney, spinal cord, and physical identifier (ear tattoo) were fixed in a 4% formaldehyde solution
- embedding media: paraplast
- thickness of sections: 5 µm
- staining: Hematoxylin and Eosin (H & E), cryo-cuts obtained from the formaldehyde -fixed livers were stained with Oil red O (ORO) and bone marrow smears were stained according to May-Gruenwald-Gimsa.
Optional endpoint(s):
Optional endpoints: No
Other examinations:
None
Statistics:
In line with the small number of animals per group, a descriptive statistical evaluation was performed. The calculations included the determination of arithmetical means and standard deviations. Minima and maxima were also given for gravimetric examinations.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 4000 ppm: reduced feed intake, refusal of feed intake after first administration, vomiting, so that after four days, animals were fed with plain diet to recover and were thereafter fed with 2000 ppm test substance in the diet for 13 weeks. After change of concentration in the diet, 1 male and 3 females were regarded as normal to thin in in Week 4, at Week 7 only 1 female was regarded as normal to thin.

Not treatment related findings were 1 female (250 ppm) appeared to be normal to thin and 1 female (1000 ppm) normal to fat at final sacrifice. There was no dose-dependency.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 4000/2000 ppm: males gained slightly less weight (-12.8%) than control animals

Summarized results can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: reduced feed intake compared to controls in Weeks 3, 4, 5 and 6 and in Weeks 10, 11, 12 and 14 (1 female), reduced feed intake compared to controls in Weeks 2 and 10 (1 female).
- 2000 ppm: When animals received 2000 ppm of the test substance, feed intake was slightly reduced in males and females compared to controls in the first half of the study.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Haematological findings:
no effects observed
Description (incidence and severity):
No striking differences were observed between control and treatment groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 250 ppm: increased GLDH in 1 female compared to controls (Week 2, seen as adaptive response)
- 1000 ppm: increased ASAT activity (1 male, Week 2) compared to controls
- 2000 ppm: increased ALAT activity in 2 males (Week 2) and 1 female (Week 2 and 7), increased GLDH for 1 male and 1 female (both Week 2), slightly increased N-Demethylase level, all compared to controls.

All changes were reversible within the study period. One male of the middle dose group showed increased ALAT activity in Week 15 compared to controls, slightly outside the +2s-range of the laboratory control values but no other liver parameters were altered, so that this finding was considered incidental. In males and females, T4 levels were slightly decreased and TSH levels increased but both the T4 and TSH values were still within the +/-2s-range of the historical control data. In the middle and high dose group, mean serum urea values were increased in Week 7 and 15 compared to controls but within the +2s-range of historical control data and considerable interindividual variation was shown in all test groups and the controls. Therefore, all findings listed were considered incidental.

Summarized data can be found in Attachment 3.

Liver enzyme activity
- 2000 ppm: slightly but statistically significantly increased EH (males), marginal but not statistically significant induction of GST and UDP-GT (females), reduced EROD (males, females), all compared to controls.

Summarized data can be found in Attachment 4.
Endocrine findings:
no effects observed
Description (incidence and severity):
see clinical biochemistry findings
Urinalysis findings:
no effects observed
Description (incidence and severity):
With respect to the parameters considered, no differences were observed between control and treatment groups.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: increased prostate weight (absolute +145.3%, relative +152.5% compared to controls)
- 2000 ppm: increased prostate weight (absolute +180.2%, relative +195.0% compared to controls)

Absolute and relative liver weight of all treatment groups was increased compared to controls (absolute +16 to + 20%, relative +18 to +22%), but no dose-relationship could be established. Liver weights of control animals were in the lower range of historical control data while liver weights of the treatment groups were in the higher range of the historical control data. Therefore, this finding is ambiguous.

Testes weight was also increased in treated groups but values were within the historical control range and the differences can be explained by different interindividual maturation stages of Beagle dogs of this age.

Summarized results can be found in Attachment 5.

Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: slight to moderate hypertrophy of the prostate (all males)
- 2000 ppm: slight to moderate hypertrophy of the prostate (all males)

In the 2000 ppm group, the following findings were not considered treatment related: The incidences of slightly increased number of degenerated spermatocytes in the testes (2/4 animals) and in the epididymides (4/4 animals - vs 1/4 control animals) and the slightly more prominent Leydig cells (3/4 animals vs 1/4 control animals) were not considered treatment related. It is known that such changes show a wide variation with respect to severity and incidence in young mature dogs.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Plasma concentrations of the test substance were measured after 13 weeks at 0, 2, 4, 6, and 24 h after feeding. The plasma concentration of the substance reached a peak after 6 h. At 0 and 24 h, the concentration were low. The Cmax values in male as in female animals were dose-proportional. In comparison to the dose the plasma levels were relatively high (relative concentration p = 0.24 - 0.36). These data showed that the test substance was absorbed to a sufficient amount.
Key result
Dose descriptor:
NOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
other: corresponding to 8.5 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 34.9 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effect observed up to the highest concentration tested.
Remarks on result:
other: corresponding to 65.3 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
male reproductive system
Organ:
other: prostate
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 6 and 7). 

Conclusions:
The present study was conducted to assess the effects of the test substance on beagle dogs when given for a time frame of approximately 13 weeks. The study followed the OECD guideline 409 (dated 1981) and was performed under GLP conditions. Beagle dogs received the test substance via the diet (dietary concentrations: 250, 1000 and 2000 ppm).

Under the conditions of the test, the test substance induced altered feed consumption and body weight in treated animals compared to controls at 1000 ppm and higher. Altered liver enzyme activity was observed, namely increased N-demethylase levels, UDP-GT and EH activity as well as reduced EROD activity in the high and - for some parameters - middle dose group. These parameters indicate increased liver metabolization. This is also seen by temporarily altered clinicochemical investigations and slightly increased liver weights at necropsy. The slight increase of UDP-GT might also explain decreased T4 and increased TSH levels. Since this can also be seen as an adaptive response and no other adverse effects were observed, it is not considered of toxicological concern. The prostate was altered both in terms of organ weight as also histopathologically in the high and middle dose group of males.
Based on these results, the NOAEL was set at 250 ppm (corresponding to 8.5 mg/kg bw/day) for male dogs and and the LOAEL was 1000 ppm (corresponding to 34.9 mg/kg bw/day). For the females, since no adverse treatment-related effects could be evidenced, the NOAEL was set at 2000 ppm (corresponding to 65.3 mg/kg bw/day), which was the highest concentration tested.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Dec 1995 - 14 Jan 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 28-33 weeks
- Weight at study initiation: 7.0-11.0 kg (males), 7.1-10.0 kg (females)
- Fasting period before study: not applicable
- Housing: individually, all animals were allowed to exercise for 60 min daily, except on weekends and public holidays.
- Diet: Ssniff HH sole diet for dog husbandry, double ground (Ssniff Versuchstierdiäten GmbH, Soest, Germany), 300 g in week 1 - 6, 330 g in week 7-17, 350 g in week 18-26, 380 g in week 27-53
- Water: tap water, ad libitum
- Acclimation period: approximately 1.5 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-23.0
- Humidity (%): 20-60
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 Jan 1996 To: 14 Jan 1997
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly, daily mixed with water to form the feed paste (see below)
- Mixing appropriate amounts with pulverized dry feed: ssniff HH sole diet for dog husbandry, double ground (ssniff Versuchstierdiäten GmbH, Soest, Germany), the dry feed was mixed with the test substance at the respective concentrations. Immediately before administration, hand-warm tap water was added to the feed meal (control) and the feed-substance mixtures at a ratio of 1:1 and a homogeneous paste was formed. This was given to the animals daily in the morning. Remaining feed that was not consumed after 1 hour was weighed.
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration (purity) of the active ingredient in the test substance was tested throughout the study by High-Performance Liquid Chromatography (HPLC). Stability and homogeneity of the test substance in the feed were tested before initiation of the study by HPLC. It was found that the test substance was stable and evenly distributed for 14 days in the dry diet (mean recovery 103.1%) and 24 hours in the moist feed (mean recovery 96.19%) within the concentration range of 20 ppm to 5000 ppm.
Additionally, the concentration of the test substance in the animal diet mixtures was verified at different time points throughout the study and found to agree with the target concentrations within defined limits. Recoveries were between 84.4 and 107.8%. Additional stability tests with these samples were conducted.
Duration of treatment / exposure:
52 weeks/26 weeks (interim sacrifice)
The last of feeding and thus final dosing with the test substance was the day prior to scheduled necropsy.
Frequency of treatment:
continuously via the diet
Dose / conc.:
40 ppm
Remarks:
main group, corresponding to 1.42 mg/kg bw/day for males and 1.39 mg/kg bw/day for females, combined 1.41 mg/kg bw/day, administered for 52 weeks
Dose / conc.:
100 ppm
Remarks:
main group, corresponding to 3.60 mg/kg bw/day for males and 3.27 mg/kg bw/day for females, combined 3.44 mg/kg bw/day, administered for 52 weeks.
Satellite group (interim sacrifice), corresponding to 3.23 mg/kg bw/day.
Dose / conc.:
250 ppm
Remarks:
main group, corresponding to 8.88 mg/kg bw/day for males and 8.30 mg/kg bw/day for females, combined to 8.58 mg/kg bw/day, administered for 52 weeks
Dose / conc.:
1 000 ppm
Remarks:
main group, corresponding to 34.42 mg/kg bw/day for males and 33.80 mg/kg bw/day for females, combined to 34.11 mg/kg bw/day.
Satellite group (interim sacrifice), corresponding to 32.21 mg/kg bw/day.
No. of animals per sex per dose:
4/sex/dose (3 males/dose for interim sacrifice)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose selection was based on the subchronic feeding study (M-003814-01-1) in beagle dogs over 13 weeks. In this study, 4 male and 4 female dogs were fed 250, 1000 and 4000 ppm of the test substance via the diet. The highest dietary concentration had to be reduced to 2000 ppm after 4 days of 4000 ppm administration and 10 days of recovery since animals started vomiting and showed reduced feed intake and body weight decrease within the first days of administration. The study duration was prolonged for 2 additional weeks.
Animals receiving 1000 and 2000 ppm of the test substance displayed signs of toxicity, mainly seen as reduced body weight and feed consumption. Hepatic enzyme induction was seen to a low extend as well as decreased T4 and increased TSH levels (slightly). Slight to moderate changes in prostate morphology and weight were observed in the middle and high dose. Plasma concentration analysis revealed high concentrations of the substance in the plasma, verifying that the substance was absorbed and distributed.
To assess whether the clinical sings seen in high dose animals (4000 ppm) were due to bad palatability, an additional subacute study was performed in which dogs were administered the test substance by capsules. The dose levels were 95 and 152 mg/kg bw/day. Severe signs of toxicity, like vomitus, reduced feed intake, increased salivation, tremor and ptosis occurred at both doses in a dose-dependent manner. 1 animal in the high dose group died and plasma concentrations on Day 1 and Day 12 showed that concentrations of the test substance on Day 12 were higher than on Day 1, indicating a building-up of the test substance.
Based on these two studies, the doses in the present study were set as follows: 0, 40, 100, 250 and 1000 ppm in the diet for 52 weeks. Additionally, groups of three male dogs received 0, 100 and 1000 ppm for 26 weeks to analyze changes in the prostate over a prolonged period of time (interim sacrifice).
Positive control:
no
Observations and examinations performed and frequency:
GENERAL OBSERVATIONS: Yes
- Time schedule: daily (repeatedly)
- observations: appearance and behavior

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of the study and in Weeks 6, 14, 26, 39 and 52
- Dose groups that were examined: all animals (before start of study and in Weeks 39 and 52), satellite groups (before start of study and in Weeks 6, 13, 26, 39 and 52)
- Parameters checked and methods used: In the ophthalmoscopic investigations the external
sections of the eye (conjunctivae, lids, sclera, cornea, lacrimal apparatus) were first examined by adspection. The transparent media (cornea, anterior chamber, iris, lens, vitreous body) and the fundus were then evaluated with the aid of the ophthalmoscope system. Mydriatic eye drops were instilled into the ventral eyelids about one hour before the start of the investigations.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of the study (Week -4, -2) and in Weeks 6, 13, 26, 39 and 52
- Anesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: all animals (before start of study and in Weeks 39 and 52), satellite groups (before start of study and in Weeks 6, 14, 26, 39 and 52)
- Method: Blood was obtained by puncturing the jugular vein. For the blood for the haematological investigations EDTA tubes (coated with dipotassium ethylene diamine tetra-acetate) were used.
- Parameters checked: differential blood count, erythrocyte morphology (= red blood cell morphology), erythrocyte count (= red blood cell count; ERY), erythrocyte sedimentation rate (ESR), hemoglobin concentration in the blood (HB) , hematocrit (= packed cell volume; HCT), Heinz bodies, leukocyte count (= white blood cell count; LEUCO), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), methemoglobin (MET-HB), reticulocytes (RETI), thrombocyte count (= platelet count; THRO), partial thromboplastin time (PTT), thromboplastin time (HepatoQuick;
HQUICK)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: Not specified
- How many animals: all animals (before start of study and in Weeks 39 and 52), satellite groups (before start of study and in Weeks 6, 14, 26, 39 and 52)
- Method: Blood was obtained by puncturing the jugular vein. For the plasma for the clinical chemistry investigations heparin-coated tubes were used, except for the electrolytes which were measured in serum and for glucose which was measured in deproteinated whole blood.
- Parameters checked: alanine aminotransferase (ALAT), alkaline phosphatase (APh), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), creatine kinase (CK), glutamate dehydrogenase (GLDH), lactat dehydrogenase (LDH), albumin (ALB), bilirubin (BILI-t), cholesterol (CHOL), creatinine (CREA), total protein (PROT), triglycerides (TRIGL), urea (UREA), glucose (GLUCOSE), chloride (Cl), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), magnesium (Mg), iron (Fe), triiodothyronine (T3), thyroxine (T4), thyroxine-binding capacity (TBC), thyroid stimulating hormone (TSH)
- Parameters checked in liver samples (collected at necropsy in Weeks 26 and 52): N- and O-Demethylase, triglycerides and Cytochrome P-450 content

URINALYSIS: Yes
- Time schedule for collection of urine: before start of the study (Week -4) and in Weeks 6, 13, 26, 39, 52
- How many animals: all animals (before start of study and in Weeks 39 and 52), satellite groups (before start of study and in Weeks 6, 13, 26, 39 and 52)
- Metabolism cages used for collection of urine: Yes (for 6 h)
- Animals fasted: Yes (during collection)
- Parameters checked: creatinine (CREA), density, sodium (Na), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:

ELECTROCARIOGRAMS (ECG)
- Time schedule: two weeks before start of treatment and in Weeks 6, 13, 26, 39, 52 (before and 2h after application)
- Animals examined: all animals (2 weeks before start of study and in Weeks 39 and 52), satellite groups (2 weeks before start of study and in Weeks 6, 13, 26, 39 and 52)
- Parameters checked and methods used: The lead electrodes were fixed subcutaneously
on the outer surface of the four extremities. The investigations were performed with an ECG recorder and evaluator manufactured by Picker International GmbH (Schwarzer CU12-RS); the standard leads I, II, III, aVR, aVL and aVF were recorded. Calibration and evaluation were automatic. The heart rates of Weeks -1, 6, 13, 26, 39 and 52 (in Weeks 39 and 52, satellite groups were not evaluated) were also measured automatically at the same time points like the ECG's.

MORPHOLOGICAL INVESTIGATION OF THE PROSTATIC GLAND
- Time schedule: Week 8, 17, 26, 35, 44, 51
- Animals examined: all male dogs (Week 35, 44 and 51), satellite groups (Week 8, 17, 26, 35, 44 and 51)
- Method: Morphological investigation including sonographic imaging

REFLEXES, BODY TEMPERATURE AND PULSE
- Parameters: pupillary, corneal, patellar, extensor, postural and flexor reflexes, body temperature and pulse frequency by palpation at the femoral artery
- Time schedule: before the start of the study (Week -4) and in Weeks 6, 13, 26, 39 and 52
- Animals examined: all animals (before start of study and in Weeks 39 and 52), satellite groups (before start of study and in Weeks 6, 13, 26, 39 and 52)


Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination under Narcoren® anesthesia.

GROSS PATHOLOGY: Yes
- Organs weighed: brain, heart, liver, lungs, spleen, adrenals, kidneys, pancreas, thyroid, pituitary, testes, prostate gland, uterus, thymus and ovaries
The diameter of the prostate was measured.


HISTOPATHOLOGY: Yes
- fixed tissues: adrenal glands, aorta, epididymides, esophagus, eyes, femur, humeri, gall bladder, heart (two papillary muscles of the left ventricle), intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, liver, lung (one lobe was instilled with 4% formaldehyde), lymph nodes (mandibular and mesenteric), optic nerves, ovaries, oviducts, pancreas, pituitary gland, prostate, salivary glands (parotid and mandibular gland), sciatic nerve, skeletal muscle (thigh), skin (mammary area with nipple), spleen, sternum, stomach (fundic and pyloric region), testes, thymus, thyroid gland with parathyroid gland, tongue, tonsils, trachea, urinary bladder, uterus, vagina and all organs/tissues with macroscopic alterations.
- lung, brain, two further liver pieces, a transversal slice of kidney, spinal cord, and physical identifier (ear tattoo) were fixed in 4%formaldehyde solution
- embedding media: paraplast
- thickness of sections: 5 µm
- staining: Hematoxylin and Eosin (H & E), cryo-cuts obtained from the formaldehyde -fixed livers were stained with Oil red O (ORO) and bone marrow smears were stained according to May-Gruenwald-Gimsa. Sections of the kidneys and livers of all animals of the terminal sacrifice groups were stained with Prussian Blue for iron and according to Schmorl to demonstrate lipofuscin.

Statistics:
In line with the small number of animals per group, a descriptive statistical evaluation was performed. The calculations included the determination of arithmetical means and standard deviations. Minima and maxima are also given for gravimetric examinations.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Main and satellite groups:
All clinical signs observed such as eczemas at the ear margin and partly also at the front paws and head or mushy and liquid feces were distributed evenly across all dose groups.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Main and satellite groups:
No differences were found between control and treatment groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Main groups:
- 40, 100 and 250 ppm: No treatment-related differences were observed between control and treatment groups up to and including 250 ppm.
- 1000 ppm: slightly reduced feed intake in females

Summarized results can be found in Attachment 1 in the attached background material.

Satellite groups:
No differences were observed between control and treatment groups.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Main and satellite groups:
No differences were found between control and treatment groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Main groups:
No differences were found between control and treatment groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main groups:
No differences were found between control and treatment groups.

Satellite groups:
There were no significant differences between control and treatment groups. One animal (1000 ppm) showed considerably reduced erythrocyte- and hemoglobin-values, accompanied by a low hematocrit. These low values led to a steep rise of the reticulocytes. This was only observed in Week 13, in Week 26 all values, despite a higher reticulocyte count, had returned to normal but mean reticulocyte counts for the whole group were elevated in Week 13 and 26 caused by this. It is assumed that the cause of this was high stress due to bleeding and it is not considered treatment related.

Individual results can be found in Attachment 2 in the attached background material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main groups:
No differences were found between control and treatment groups. Individually, in the 250 and 1000 ppm group, ALAT, GLDH, ASAT and LDH activities were slightly increased but within the biological variation and none of these findings was seen in Week 52 anymore. One animal (1000 ppm) had increased CK activity, probably resembling some externally not visible muscle injury/trauma, leading also to increased ASAT, ALAT and LDH levels. In liver tissues, one animal (100 ppm) had increased triglyceride values which caused an increased mean triglyceride value in this group. None of these findings were considered treatment related.

Satellite groups:
At 100 and 1000 ppm, T4 levels were slightly decreased but within the historical control range (Attachment 6 in the attached background material). In 1 animal (100 ppm), increased CK- and LDH values occurred in Week 13 but returned to normal in Week 26 and this was not seen in any other animal. Therefore, this finding was considered incidental.

Individual results can be found in Attachment 2 in the attached background material, T3 and T4 values are listed in Attachment 3 and reference values in Attachment 6 in the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
Main and satellite groups:
No differences were observed between control and treatment groups.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Necropsy in Week 52 (main groups):
- 40, 100 and 250 ppm: No treatment-related differences were observed between control and treatment groups up to and including 250 ppm.
- 1000 ppm: prostate weight was slightly increased (due to extremely high prostate weight of one animal) but variation was also high (as seen as high values for control animals), so that this finding is ambiguous.

Summarized data can be found in Attachment 4.

Necropsy in Week 26 (satellite groups):
Prostate: one animal in the control and one animal of the 100 ppm group had rather low absolute prostate weights which was considered incidental. Apart from that, no differences were found.
Liver: In the 1000 ppm group (males), absolute and relative liver weights were slightly increased but within the historical control data.
Kidney: Increased absolute and relative kidney weight in the 100 and 1000 ppm group but within the historical control range

Summarized data can be found in Attachment 5 in the attached background material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy in Week 52 (main groups):
No differences were found between control and treatment groups up to and including 100 ppm.
- 1000 ppm: The prostate was slightly enlarged in the high dose group.

Necropsy in Week 26 (satellite groups):
No differences were found between control and treatment groups.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necropsy in Week 52 (main groups):
No treatment related effects were observed up to 1000 ppm in both sexes. All variations were evenly distributed among the treatment groups. One female of the high dose group had a moderately increased iron storage in Kupffer cells, periportal macrophages and hepatocytes but this was not found in any other animal. Also, no effects on iron metabolism, hematopoiesis and red blood cell degradation were found in any study with the test compound, so that this finding was considered incidental.

Necropsy in Week 26 (satellite groups):
- 100 ppm: No treatment-related differences were observed between control and treatment group.
- 1000 ppm: hepatocellular cytoplasmic change (males) in line with the increased liver weights observed in the high-dose males at interim sacrifice.
At terminal sacrifice (52 weeks) this finding was not observed. Therefore, this finding was regarded as a temporary adaptive response to the test substance administration and metabolism. It was therefore not regarded as a toxicologically relevant adverse effect.


Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no neoplastic changes observed in any dose or control group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Sonographic prostate examinations: effects observed, not treatment-related
- 40 ppm: maximum width of the left lobe measured in transverse plane differed in Week 51 compared to controls
- 250 ppm: maximum width of the left lobe measured in transverse plane differed in Week 17 and 26 and in Week 51 compared to controls, maximum width of the right lobe in transverse plane differed to controls in Week 17
- 1000 ppm: maximum width of the left lobe measured in transverse plane differed in Week 51 compared to controls

All these findings were within the biological variation and related to biological differences in the maturation development and were therefore not considered treatment related.

- Reflexes: Tests of the reflexes did not yield any pathological findings at any time or in any dose group.
- Pulse rates: The pulse rates showed no toxicological relevant alterations up to and including 1000 ppm.
- Body temperature: The body temperatures showed no toxicological relevant alterations up to and including 1000 ppm.
- Heart rates: Heart rates did not yield any striking differences between the controls and the animals in the treatment groups.
- Electrocardiograms: The electrocardiograms showed no toxicological relevant alterations up to and including 1000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: corresponding to 8.88 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 34.42 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effect observed at this dose level.
Remarks on result:
other: corresponding to 33.8 mg/kg bw/day
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 ppm

Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 4 and 5). 

Conclusions:
The present study was conducted to assess the effects of the test substance on beagle dogs when given for a time frame of approximately 52 weeks. The study followed the OECD guideline 452 and was performed under GLP conditions. It was therefore considered valid and reliable.

The test item caused a slight change of feed intake (females) and liver weight (males) in the highest dose groups after 52 or 26 weeks of administration, respectively. The weights and size of the prostate gland were slightly increased in the male dogs at 1000 ppm but without any histopathological or sonographical correlate. Also clinicochemical investigations did not show effects on hepatic enzyme induction or thyroid impairments as seen in rodent studies. Therefore, a dose level of 250 ppm (corresponding to 8.88 mg/kg bw/day) is regarded as the NOAEL for dietary administration of the test substance to the beagle dog over 52 weeks in males and in females the NOAEL was set at >= 1000 ppm (corresponding to 33.80 mg/kg bw/day).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Apr - 15 Aug 1994
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)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Mainly missing examinations, for details please refer to "Principles of method if other then guideline"
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
Animals were grouped individually, no sensory reactivity was assessed, no lipid analysis in blood, it was not specified if the brain was sectioned for histopathology, endocrine parameters and terminal vaginal cytology were not investigated, various organs were not weighed: pituitary gland, spleen, thymus, uterus, prostate, the estrus cycle was not determined at necropsy.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Common strain for toxicological studies and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 5 - 6 weeks
- Weight at study initiation: 78-113 g (males), 77-102 g (females)
- Fasting period before study: not applicable
- Housing: individually under conventional conditions in type IIa polycarbonate cages on low-dust wood granulate (type S 8/15, S sniff Spezialdiäten GmbH, Soest, Germany), during acclimatization rats were kept in groups (about 5 animals per type III cage, separated by sex)
- Diet: fixed-formula standard diet (Altromin® 1321 meal supplied by Altromin GmbH, Lage,
Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet (Altromin® 1321 meal supplied by Altromin GmbH, Lage, Germany). The test substance was blended (using a mixing granulator, Loedige, Paderborn, Germany) with Altromin 1321 meal. To all diet mixtures including the control, peanut oil (DAB 10) was added to minimize dust formation.
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: peanut oil (DAB 10) was mixed in the feed to prevent dust-formation
- Amount of vehicle in feed: 1%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on homogeneity and stability of the test substance in the administration vehicle covering the dose range used were obtained. It was found that the test substance was stable in the diet for 8 days (96-105% recovery).
Samples for the homogeneity analyses were obtained before start of the study (10 and 20000 ppm). Homogeneity of test substance distribution in the feed was confirmed at 10 and 20000 ppm (nominal) as the standard deviation of the analytical results did not exceed 10% in six samples of each concentration.
For verification of doses, three times throughout the study period, samples of the actual feed mixes were taken at the nominal concentrations in feed of 25, 100, 400 and 1600 ppm. They were collected on the day of preparing the feed mixture and after a storage period of 7 or 10 days under animal room conditions. Mean recoveries were 96.3, 106, 106 and 101%, respectively. The mean analytically verified concentrations of the test substance in feed were 24.1, 106, 424 and 1610 ppm.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
continuously via the diet
Dose / conc.:
25 ppm
Remarks:
corresponding to 1.9 and 2.0 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
100 ppm
Remarks:
corresponding to 7.3 and 7.6 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
400 ppm
Remarks:
corresponding to 28.6 and 35.6 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
1 600 ppm
Remarks:
corresponding to 123.2 and 160.6 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
10 (main dose and satellite groups)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were based on a previously conducted subacute study with the test substance in which doses of 0, 5, 20, 60 and 120 mg/kg bw/day were tested. All test material treatments resulted in increased liver enzyme activity. From 20 mg/kg bw/day onward, water consumption was increased, cell counts in lymph nodes were reduced, and an increase in macrophage activation was observed. 60 mg/kg bw/day and higher doses led to decreased body weight, feed consumption and leucocyte counts. In the high dose group, animals showed decreased reactivity, slight effects in the liver were found, increased concentrations of inorganic phosphate, reduced cell counts in spleen and increased cell proliferation in liver tissue were observed additionally. Based on these results, 25, 100, 400 and 1600 ppm were selected as dose levels.
- Rationale for selecting satellite groups: recovery after treatment. The satellite groups were fed the control diet for an additional observation period of 5 weeks after completion of the regular exposure period of 13 weeks (0 and 1600 ppm).
- Post-exposure recovery period in satellite groups: 5 weeks
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on holidays and weekends)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- observations: Body surfaces and orifices, posture, general behavior, breathing and excretory products

BODY WEIGHT: Yes
- Time schedule for examinations: before beginning of the study and weekly thereafter and directly before necropsy


FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at start of the study (all groups), Week 12 (all animals of main control and high dose group)
- parameters examined: pupillary reflex of both eyes was tested in a darkened room and inspection of the anterior regions of the eye was performed, after that, pupils were dilated with Mydriaticum Roche® or Mydriaticum Stulln® drops, the refractive elements of the eye as well as the fundus were examined using an indirect ophthalmoscope and a ZEISS photo-slit lamp.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 3 and 11/12 (main groups) and 17 (recovery groups)
- Anaesthetic used for blood collection: No (for glucose concentration, blood was taken in the morning from caudal veins) and yes (with ether, for determining the other parameters in peripheral blood, blood was collected in the morning from the retro-orbital venous plexus)
- Animals fasted: No
- How many animals: all animals
- Parameters checked: differential blood count including lymphocytes (LYM), segmented neutrophils (SEGM), eosinophils (EOS), monocytes (MONO) and band neutrophils (BAND), morphology of erythrocytes, erythrocytes (ERY), Heinz bodies (HEINZ), haemoglobin (HB), haematocrit (HCT), leucocytes (LEUCO), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet count (THRO), hepato quick (HQUICK), reticulocytes (RETI).

CLINICAL CHEMISTRY: Yes
- Time schedule and procedure for collection of blood: see haematology
- How many animals: all animals
- Parameters checked: alkaline phosphatase (APh), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), glutamate dehydrogenase (GGT), albumin, protein (total), bilirubin (total, BILI-T), cholesterol (CHOL), creatinine (CREA), glucose (GLUC), triglycerides, urea, calcium, chloride, potassium, sodium, phosphorus, triiodothyronine (T3), thyroxine (total, T4), thyroxine-binding capacity (TBC)
- Liver tissue was sampled from 5 rats per sex and group at necropsy of main and recovery groups and checked for N-demethylase, O-demethylase, cytochrome P-450, triglycerides, 7-ethoxycoumarin deethylase (ECOD), 7-ethoxy-resorufin deethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), Glutathione-S-transferase (GST), UDP-Glucuronyl-transferase (UDP-GT)

URINALYSIS: Yes
- Time schedule for collection of urine: Week 3 and 11/12 (main groups) and 17 (recovery groups); collection period of approx. 16 hours
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, the rats were administered 20 mL/kg bw water by stomach tube before urine collection. No feed or water was provided during the collection period.
- Parameters checked: specific gravity of urine, calcium, potassium, sodium, volume, bilirubin, blood, glucose, ketone bodies, pH-value, protein, urobilinogen, urine sediment

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: Yes
- Time schedule for examinations: necropsy at end of treatment and recovery period
- How many animals: 5 per sex and group
- Dose groups that were examined: all (main and recovery)
- tissues sampled: blood, mesenteric lymph nodes, femur with bone marrow and half the spleen
- Parameters checked: cell counts, FACScan-analyses, macrophage activity after PMA stimulation,
mitogen stimulation and antibody titers in serum

OTHER: Cell proliferation was investigated in kidney and liver samples that were collected at necropsy of main and recovery groups.
Sacrifice and pathology:
At the end of the treatment or the recovery phase, surviving animals were sacrificed with exsanguination under deep ether anesthesia and submitted to necropsy.

GROSS PATHOLOGY: Yes
For the following organs, weights were recorded at necropsy: brain, heart, liver, lung, spleen, kidneys, adrenal glands, thyroid, testes and ovaries.

HISTOPATHOLOGY: Yes
- tissues collected and fixed in Bouin's solution: aorta, adrenal glands, bone marrow (in femur and sternum), brain (in toto), caecum, colon, duodenum, epididymides, extraorbital lacrymal glands, eyelids, eyes, femur with knee joint, harderian glands, head, heart, ileum, jejunum, kidneys, larynx, liver*, lungs**, lymph nodes (mesenteric and mandibular), mammary glands, musculature (femoral), oesophagus, optic nerve, ovaries, oviducts, pancreas, pituitary gland, prostate, rectum, residual intestine, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord (vertebral column: cervical, thoracic, lumbar), spleen, sternum, stomach, tattooed auricles, testes, thymus (if present), thyroid + parathyroid, tongue, trachea, urinary bladder***, ureter, urethra, uterus, vagina, zymbal glands
* one lobe was fixed in 10% buffered formaldehyde solution, one lobe was retained and deep frozen for biochemical investigations
** lungs fixed by instillation with 5% and then fixed in 10% buffered formaldehyde solution
*** bladder fixed by instillation
- embedding media: paraffin (with the exception of esophagus, eyelids, head, larynx, remaining intestinal tissues, trachea, ureter, urethra and physical identifier)

Staining: hematoxylin and eosin (H&E)
- dose groups: all (livers, lungs, kidneys and thyroid glands), or only control and high dose animals (all other organs)

Staining: Oil Red 0 (ORO)
- tissues: liver cryocuts (for lipid determination)
- dose groups: all
Optional endpoint(s):
no
Other examinations:
no
Statistics:
The statistical evaluation of data related to clinical chemistry, haematology, survival, body and organ weights as well as feed and water intake was performed using SAS® routines.

For continuous data presumed to be normally distributed with equal variances, an ANOVA followed by the Dunnett test was used. If heteroscedasticity appeared more likely a p value adjusted Welch test is applied. If no parametric analysis could be done, Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon (U tests) was performed.

For global tests, multiple comparison procedures were used (two-sided).

Significant differences from the control group were indicated with "+" for p <= 0.05 and "++" for p <= 0.01. In case of numbers of values too low to calculate test statistics this was indicated by "0".
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: transient pallor, distended abdomen, tilted head, labored breathing and narrowed eyelids (1 male)

Not treatment related:
- Control: transient eye opacity (1 female)
- in all dose groups: occasionally and sporadically, hair loss occurred (males/females)
Mortality:
no mortality observed
Description (incidence):
No premature deaths occurred during this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Main group:
- 25, 100 and 400 ppm: There was no effect of treatment on body weight development in the main groups up to and including 400 ppm.
- 1600 ppm: lower body weights and retarded body weight gain (males, females), statistically significantly decreased compared to controls until end of the study, terminal body weight was reduced in males (-16%) and females (-14%).

Recovery groups:
- 1600 ppm: lower body weights and retarded body weight gain (males, females), statistically significantly decreased compared to controls until end of the treatment period, during recovery the difference remained but diminished compared to controls.

Summarized data can be found in Attachment 1in the attached background material.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences between control and treatment groups (both main and recovery groups) were observed.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 25, 100 and 400 ppm: There was no effect of treatment on water consumption in the main groups up to and including 400 ppm.
- 1600 ppm: average total and daily water consumption per animal was reduced compared to controls (males)

Not treatment related:
In females of the high dose group, water consumption was higher in g per animal/day and per kg bw/day than in controls but total and daily water consumption did not show a dose-related tendency, so that the finding was considered not of toxicological relevance.

Summarized data can be found in Attachment 2 in the attached background material.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups (both main and recovery groups) were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):

- 1600 ppm: in Week 3 and 12, mean clotting times were shorter (females)

Not treatment related:
All comparisons were made to controls. At 1600 ppm, erythrocyte counts in males were slightly increased at one time point but were within the range of historical control data (Attachment 11) and the finding was therefore not considered of toxicological relevance. In terms of leucocyte count, different deviations from the control group were found throughout the treatment period but were not considered biologically relevant, since no dose-dependency was established, they were within the normal range of variation of historical control data (Attachment 11), the deviations occurred only at one time point and/or in one sex and/or after the end of treatment in the recovery group. The only observation that can be seen as ambiguous is the reduced monocyte count. In females, the monocyte count is reduced in Week 12 from 100 ppm onward. The data for 100, 400 and 1600 ppm show statistical significance but are not dose-dependent, since the 1600 ppm result is higher than the 400 ppm results. Moreover, in males, a dose-dependent decrease is observable in Week 12 but none of the results was statistically significant.

No differences attributable to treatment were observed at any time in males and females at doses up to and including 1600 ppm regarding erythrocyte parameters and morphology, hemoglobin concentration and hematocrit values.

Summarized data can be found in Attachment 3 and 4 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 25 and 100 ppm: No differences regarding clinical biochemistry were observed between control and treatment groups up to and including 100 ppm.
- 400 ppm: increased cholesterol and protein concentration (males, females) compared to controls.
- 1600 ppm: increased cholesterol and protein concentration (males, females), increased creatinine in males in Week 12, both compared to controls.

All findings were reversible. All other occurrences of statistical significance were not dose related, minimal and occurred only sporadically. They were therefore not considered biologically significant.

Thyroidal hormones
- 25 and 100 ppm: No differences regarding thyroidal hormones were observed between control and treatment groups up to and including 100 ppm.
- 400 ppm: increased T4 in Week 3 (males), increased T3 levels (males, Week 3), all compared to controls.
- 1600 ppm: increased T3 in males at 3 and 12 Weeks, compared to controls.

All findings were reversible.

The thyroxine-binding capacities (TBC) were increased in males of all dose levels in Week 12, and in the high dose in Week 3. In females, TBC was increased in Week 3 in the 400 and 1600 ppm dose groups. Even though the increases were statistically significant, they were not considered toxicologically relevant, since they were small and the individual animal data lay mostly within the range of reference values for animals of that age. The only observation that was outside the range of reference values was the increase of TBC in individual animals of the 100 ppm treatment group (males), but since this was an isolated finding, it was not considered of biological relevance.

Also, T3 increased in males in Week 3 already at 25 ppm showing statistical significance. However, the increase was small and not seen in Week 12, where only treatment with 1600 ppm led to a statistically significant increase. Therefore, this finding is considered ambiguous.

Summarized data can be found in Attachment 6 in the attached background material.

Liver tissues
- 25 and 100 ppm: No differences regarding clinical biochemistry in liver tissue were observed between control and treatment groups up to and including 100 ppm.
- 400 ppm: increased N-demethylase (males, females), O-demethylase (males), P 450 (males, females), all compared to controls.
- 1600 ppm: increased N-demethylase (males, females), O-demethylase (males, females), P 450 (males, females), increase in ECOD, EROD, ALD (cytochrome dependent monooxygenases), EH, GST and UDP-GT (males, females), all compared to controls.

At 100 ppm, slightly increased cytochrome P-450 levels were found in males compared to controls. These differences were small and within the historical control range (Attachment 11) but in line with the increased cytochrome P 450 levels at 400 and 1600 ppm, so that this finding is considered ambiguous. At 400 ppm, an increase in cytochrome dependent monooxygenases, EH, GST and UDP-GT was observed compared to controls but did not yet exceed a factor of 2.

All effects were reversible untile the end of the recovery period.

Summarized data can be found in Attachment 7 in the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 25, 100 and 400 ppm: No differences regarding urinalysis between control and treatment groups were observed up to and including 400 ppm.
- 1600 ppm: increase of urine sodium and urine calcium (males, Weeks 3, 11/12, 17) compared to controls.

Summarized data can be found in Attachment 8 in the attached background material.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
- 25, 100 and 400 ppm: No differences regarding immunological findings were observed between control and treatment groups up to and including 400 ppm.
- 1600 ppm: slight macrophage activation in the spleen (males, females), increase of the lipopolysaccharide (LPS) stimulated spleen cells (males), all compared to controls.

No treatment-related changes in antbody titers were observed (IgA, IgG, IgM). Cell counts of the lymph nodes, spleen and bone marrow did not indicate a treatment-related effect.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 25, 100 and 400 ppm: No toxicologically relevant differences regarding organ weights were observed between control and treatment groups up to and including 400 ppm.
- 1600 ppm: increased liver weights (males, females) and thyroid weights (males) compared to controls.

Increased liver weight was already visible in males treated with 400 ppm for 13 weeks, however this finding was only statistically significant compared to controls relative to body weight. Absolute liver weights were slightly increased but without reaching statistical significance.
Due to the lower body weights of the male rats treated at 1600 ppm for 13 weeks, the absolute organ weights were lower than the respective control weights.
Other non-treatment related findings were due to the lower body weights (higher relative organ weights of testes, and brain in males as well as spleen and brain in females, both at 1600 ppm).

All findings were reversible.

Summarized data can be found in Attachment 9 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
no differences between control and treatment groups (main and recovery)
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 25 and 100 ppm: No differences regarding histopathological findings were observed between control and treatment groups up to and including 100 ppm.
- 400 ppm: moderate hepatocellular hypertrophy associated with fine granular to vesicular structure in the perinuclear cytoplasm (9/10 males and 2/10 females in the main groups)
- 1600 ppm: moderate hepatocellular hypertrophy associated with fine granular to vesicular structure in the perinuclear cytoplasm (all males and females of the main group), minimal hepatocellular hypertrophy without cytoplasmic change (3/10 males in recovery group)

Summarized reuslts can be found in Attachment 10 in the attached background material.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplasticity was observed.
Other effects:
no effects observed
Description (incidence and severity):
In the assessment of cell proliferation, no differences were found between control and treatment groups.
Key result
Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 7.3 mg/kg bw/day for males and and 7.6 mg/kg bw/day females
Key result
Dose descriptor:
LOAEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
histopathology: non-neoplastic
immunology
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake
Remarks on result:
other: corresponding to 28.6 mg/kg bw/day for males and 35.6 mg/kg bw/day for females
Key result
Critical effects observed:
no
Conclusions:
This study was conducted according to OECD Guideline 408 with the following deviations: the animals were grouped individually, several examinations were not performed such as sensory reactivity, endocrine parameters, lipid analysis in blood and only few organs were weighed at necropsy. It was performed under GLP conditions and is considered valid. Treatment with the test substance led to decreased body weight and body weight gain in males and females treated with the highest dose of 1600 ppm in the diet. The difference in body weight decreased but was still observable at the end of the recovery period. Males of the 1600 ppm group showed lower water consumption; in females of this group, hematological investigations revealed a shorter mean clotting time. The test substance had an influence on the liver at 400 ppm and higher doses as seen by histopathological findings (moderate hepatocellular hypertrophy associated with perinuclear cytoplasm) and increased liver weights in males and females. Hepatic enzymes also increased in males and females at 400 ppm and higher doses (Phase I- and Phase II-enzyme activities, cytochrome P-450 levels and triglyceride concentrations). The histopathological findings were partly and the enzyme activities and macropathological findings fully reversible.
Thyroid effects were only observed in males. Triiodothyronine (T3) and thyroxine-binding
capacity (TBC) were increased in the high dose group as well as the thyroid weight. Effects at lower doses were not considered relevant since they were in the range of historical control data. The change of thyroidal parameters is considered a secondary effect to hepatic enzyme induction, especially UDP-glucuronyl-transferase (UDP-GT), and higher thyroxine (T4) turnover.
The higher protein (males from 400 ppm, females from 100 ppm onward) and cholesterol blood concentrations (males and females at 400 ppm and higher doses) in Week 3 could be considered a sign of transient effects on fat and protein metabolism. However, this was not confirmed by any other finding. All findings on thyroid parameters were reversible within the recovery period.
Urinalysis did not indicate severe kidney damage, the only finding was increased urine sodium and calcium in males receiving 1600 ppm of the test substance.
Ophthalmological and histopathological investigations provided no evidence of any toxic effects on the eye at the doses investigated. Moreover, no other organs seemed to be affected by the test substance.
Regarding the immune system, slightly increased macrophage activation, and the increase of mitogen stimulation (increase of the LPS stimulated cells) in the spleen at 1600 ppm was considered test-substance related but a secondary effect due to other organ-influencing effects of the test substance.

Taken together, subchronic administration of the test substance via the diet resulted in toxicologically relevant effects in rats at 400 ppm and higher doses. Therefore, the NOAEL was set at 100 ppm (corresponding to 7.3 and 7.6 mg/kg bw/day for males and females, respectively). In the rat, the liver was identified as the main target organ after sub-chronic dietary administration. But although there were changes in some clinical chemistry parameters (liver enzymes and thyroid hormones) at dose levels potentially relevant for classification according to CLP Regulation, these changes are considered indicative of increased liver and thyroid activity as the result of adaptive changes. Such adaptive responses are not considered adverse changes indicative of specific target organ toxicity relevant for CLP classification.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar 1995 - 7 Apr 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Mainly missing examinations, namely blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology, the feed was not analyzed for phytoestrogens.
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used for toxicology testing and recommended by the guideline
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 94-141g (males), 79-113 g (females)
- Fasting period before study: not applicable
- Housing: individually under conventional conditions in type IIa polycarbonate cages on low-dust wood granulate (S sniff Spezialdiäten GmbH, Soest, Germany)
- Diet: fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 8 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly checked for contaminations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Mar 1995 To: 7 Apr 1997
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany). The test substance was blended (using a mixing granulator) with Altromin 1321 meal. To all diet mixtures including the control, peanut oil (DAB 19, 10 g per kg feed) was added to minimize dust formation.
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: peanut oil (DAB 10) was mixed in the feed to prevent dust-formation
- Amount of vehicle in feed: 1%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on homogeneity and stability of the test substance in the administration vehicle covering the concentration range used were obtained before start of this study and homogeneity and stability were verified within a concentration range of 10 ppm to 20000 ppm. Under the sample preparation and handling conditions employed, stability in the diet was assured for a period of at least 14 days.

The test substance content of the diet mixtures was checked several times during the study (at least at start and end of the study, and several times during the study). For that purpose, samples of food mixes per dose were taken at the start of the feeding period and again at the end of the period after being kept under animal room conditions. All samples taken were kept deep frozen (<= -15°C) until examination.
Analyses were conducted using a HPLC method.
The test material content in the diet mixtures fed to the animals, prepared during the study, agreed with the target concentrations within defined limits. Recovery was between 48 and 137% for the 25 ppm samples, between 60 and 120% for the 50 ppm samples, beween 88 and 114% for the 500 ppm samples, and between 83 and 113% for the 1000 ppm samples. As the recoveries determined for the two lower concentrations (25 and 50 ppm) were in some cases not valid, the procedure was adapted. Measurements using the new procedure were in agreement with the target concentrations.
Duration of treatment / exposure:
107 weeks (54-55 weeks for interim sacrifices)
Frequency of treatment:
continously via the diet
Dose / conc.:
25 ppm
Remarks:
corresponding to 1.2 and 1.6 mg/kg bw/day actual dose ingested (males and females, respectively)
Dose / conc.:
50 ppm
Remarks:
corresponding to 2.5 and 3.3 mg/kg bw/day actual dose ingested (males and females, respectively)
Dose / conc.:
500 ppm
Remarks:
corresponding to 25.2 and 33.5 mg/kg bw/day actual dose ingested (males and females, respectively)
Dose / conc.:
1 000 ppm
Remarks:
corresponding to 51.7 and 69.1 mg/kg bw/day actual dose ingested (males and females, respectively)
No. of animals per sex per dose:
50 / 10 (interim sacrifice after 1 year)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose selection was based on results of a previously conducted subchronic toxicity study in Wistar rats (M-000863-01-1) in which rats received 0, 25, 100, 400 or 1600 ppm of the test substance in the diet. Body weight and body weight gain was reduced in 1600 ppm males and females. At 400 and 1600 ppm, liver enzymes were induced markedly, resulting in histopathological and macropathological changes (moderate hepatocellular hypertrophy). Therefore, 0, 25, 500 and 1000 ppm were administered in this study.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: A detailed weekly report on the condition of the individual animals assessed the following: body surfaces and orifices, posture, general behavior, breathing and excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: Week 0-13 weekly; Week 14-105 every two weeks. Furthermore, body weights were recorded immediately before scheduled necropsy, for calculation of relative organ weights.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations: Week 1-13 weekly; Week 14-105 every 4 weeks

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Week 1-13 weekly; Week 14-105 every 4 weeks

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment (Week 0), after 12 months and at the end of treatment (Week 103/105)
- Dose groups that were examined: pre-treatment and before necropsy after 2 years (all animals), after 1 year (control and highest treatment group)
- parameters checked: pupillary reflex of both eyes was first tested in a darkened room. After dilating the pupils with Mydriaticum-Stulln® drops, the refractive elements of the eye and the fundus were examined using an indirect ophthalmoscope. The eyes were also examined using a photo-slit lamp.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 26/29* (*when samples from Week 26 were found to be partly clotted, they were replaced with samples from Week 29), 53, 78 and 105
- Anaesthetic used for blood collection: No (for blood withdrawl from the caudal veins for measurement of glucose concentration) Yes (diethyl ether, for blood withdrawl from the retro-orbital venous plexus for measurement of other parameters)
- Animals fasted: No
- How many animals: 10 anmials per group
- Parameters checked: differential blood count, erythrocyte morphology (= red blood cell morphology), erythrocyte count (= red blood cell count; ERY), hemoglobin concentration in the blood (HB), hematocrit (= packed cell volume; HCT), leukocyte count (= white blood cell count; LEUCO), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thrombocyte count (= platelet count; THRO), thromboplastin time (Hepato-Quick; HQUICK)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 26, 53, 78 and 105
- Animals fasted: No
- How many animals: 10 animals per group
- Parameters checked alanine aminotransferase (ALAT), alkaline phosphatase (APh), aspartate aminotransferase (ASAT), gamma-glutamyltransferase (GGT), albumin (ALB), bilirubin (BILI-t), cholesterol (CHOL), creatinine (CREA), total protein (PROT), triglycerides (TRIGL), urea (UREA), glucose (GLUCOSE), chloride (Cl), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), triiodothyronine (T3), thyroxine (T4), thyroxine-binding capacity (TBC), thyroid stimulating hormone (TSH)
- Time schedule for liver investigations: Weeks 54/55
- How many animals: 10 rats/sex at 0 and 25 ppm and 5 rats/sex at 50, 500 and 1000 ppm.
- parameters checked: Cytochrome P-450 monooxygenases: 7-ethoxycoumarin-deethylase (ECOD), 7-ethoxyresorufin-deethylase (EROD), aldrin-epoxidase (ALD); Phase Il-enzymes: epoxide hydrolase (EH), glutathione S-transferase (GS-T), UDP-Glucuronyl-transferase (UDP-GT)

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 25, 52, 77 and 104 (over 16 h)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes, but water was supplied
- parameters checked: blood, bilirubin, glucose, ketone bodies, pH, protein, sediment, urobilinogen, protein (PROT), volume (VOL), calcium (Ca), inorganic phosphate (P), potassium (K), sodium (Na), density

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
All animals were killed by exsanguination under diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Animals that died spontaneously or were sacrificed in a moribund state during the study, were dissected at the earliest opportunity.

GROSS PATHOLOGY: Yes

Organ weights were recorded for brain, heart, liver, spleen, kidneys (both), adrenal glands (both), ovaries and testes (both)


HISTOPATHOLOGY: Yes
- fixed organs: Adrenals, aorta, brain (cerebrum, cerebellum, pons/medulla), cecum, colon, duodenum, epididymides, esophagus, eyes (with eyelids), exorbital lacrimal glands, femur (incl. bone marrow and knee joint), harderian glands, head-nose-pharynx area, heart, ileum, jejunum, kidneys, larynx, liver, lungs (prefixation by instillation with 4% buffered formaldehyde solution), lymph nodes (mandibular and mesenteric), mammary glands, optic nerves, ovaries (incl. oviduct), pancreas, physical Identifier (tattooed ears), pituitary, prostate, rectum, remaining, intestine, salivary glands, sciatic nerve, seminal vesicles (with coagulating glands), skeletal muscle, skin (mammary region), spinal cord (cervical, thoracal, lumbar), spleen, sternum (with bone marrow), stomach (forestomach and glandular stomach), testes, thymus (if present), thyroid (with parathyroids), tongue, trachea, ureter, urethra, urinary bladder (prefixation by instillation with 4% buffered formaldehyde solution), uterus (with cervix), vagina, Zymbal glands and all tissues showing abnormalities

Staining: hematoxylin and eosin (H&E)
- embedding media: paraplast
- tissues: all organs except eyelids, head, larynx, Zymbal glands, remaining intestinal tissue, ureters, urethra, and physical identifier
- section thickness: 5 µm (4 µm for the uterus of the intermediate group females)
- animals investigated: All main groups were examined histopathologically (all organs listed above from all animals of all dose groups).
From the animals scheduled for interim sacrifice all blocked organs were examined in control and high-dose group. In the remaining groups after interim sacrifice, only the following organs were examined: lungs, liver kidneys, heart, thyroids, parathyroids, esophagus, trachea, and all gross findings.


Staining: Oil Red O
- animals: interim sacrifice animals
- tissues: cryocuts obtained from the formalin-fixed livers

In an ammendment to the report, the uterus (horns and cervix) of the 25 and 500 ppm interim groups were also evaluated histopathologically.
Optional endpoint(s):
no
Statistics:
The statistical evaluation of data related to clinical chemistry, hematology, survival, body and organ weights as well as feed and water intake was performed using SAS® routines.

For continuous data that is presumed to be normally distributed with equal variances, an ANOVA followed by the Dunnett test was used. If heteroscedasticity appeared more likely a p value adjusted Welch test was applied. If no parametric analysis could be done, Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon (U tests) was performed.

For global tests, multiple comparison procedures were used (two-sided).

Significant differences from the control group are indicated with "+" for p < 0.05 and "++" for p < 0.01.
Clinical signs:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the course of the study, no evidence of a test substance-related increase in mortality was found. The incidences of death in control females was rather high. Cumulative mortalities were 11-12-14-10-9 and 28-22-14-19-17 for males and females, respectively (0-25-50-500-1000 ppm).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
25 and 50 ppm: There were no effects on body weights observed up to and including 50 ppm in males and females.
- 500 ppm: during the first 10 weeks of the study body weights in males were statistically significantly lower than in the corresponding control (males, Week 1-10, up to -7%), statistically significantly lower body weights than controls were observed in females starting at week 8 (continued throughout the study, up to -15%).
- 1000 ppm: statistically significantly reduced body weight in males and females to the control, in males the difference was lower and fluctuated between -5% and - 12% throughout the study, with -9% at termination, in females reduced body weight became statistically significant in Week 5 and stayed reduced throughout the study (up to -21%), all compared to controls.

Summarized data can be found in Attachment 1.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 25 and 50 ppm: There were no effects on food consumption observed up to and including 50 ppm in males and females.
- 500 ppm: food consumption was reduced at several intervals during the study (slightly but statistically significantly) compared to controls.
- 1000 ppm: food consumption was reduced at several intervals during the study (slightly but statistically significantly) compared to controls.

Summarized Data can be found in Attachment 2 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No differences between control and treatment groups were observed.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Interim examination: No differences were found after one year between control and treatment groups.

Terminal examination:
- 1000 ppm: higher incidence of lens alterations in the cortex/nucleus (females) compared to controls.

Not treatment related findings
At 50 ppm and above, males showed increased cortical water clefts. Cortical or nuclear lens opacity incidences of all degrees were found in males in all treatment groups. However, both of these signs are common in aging rats. There was no dose-dependency observed when assessing both lesions together (17-31-37-23-23%). Therefore, these lesions were not considered treatment related. The incidence of lens alterations in the cortex/nucleus in females at 25 ppm was already higher than controls (incidence 2-15-13-18-32%). However, the controls were very low which could be due to the fact that the survival rate in control females was lower than in treatment groups. Moreover, up to 500 ppm, no dose-dependency could be established and the values were still within biological variance. Therefore, only for the high-dose females a treatment-related effect was considered. Similarily, the incidences of opacities in the retrolenticular area were increased in treatment groups of 50 ppm and higher in females (incidences 2-0-13-11-14%) but control and 25 ppm values were very low, no clear dose-dependency was found and males exhibited equally distributed incidences at higher degrees (26-27-20-25-26%). Therefore, this finding was not considered treatment-related.

Summarized data can be found in Attachment 3 in the attached background material.
Haematological findings:
no effects observed
Description (incidence and severity):
Several values were statistically significantly different to control values at different time points, but no dose- or time-dependency could be established and/or the difference was not of biological significance.

Summarized data can be found in Attachment 4 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 25 and 50 ppm: There were no effects on clinical biochemistry observed up to and including 50 ppm in males and females.
- 500 ppm: slightly but statistically significant decreased triglycerides (females, all dates), slightly decreased total bilirubin (males, statistically significant at all dates), all compared to controls.
- 1000 ppm: statistically significant findings were decreased ASAT and APh (in Week 53, males), slightly increased cholesterol (Week 26 in males, females), slightly decreased triglycerides (females, all dates), slightly decreased total bilirubin (males, all dates), increased total protein (males and females, all dates), all compared to controls.

In blood electrolyte concentrations, no toxicologically relevant differences occurred. If values differed significantly from controls, these findings were sporadic, not dose-dependent and/or within the biological deviation.

Thyroid parameters:
- 500 ppm: increased TSH in some males (Week 26) compared to controls.
- 1000 ppm: non-statistically significant and mostly slight increase in the TSH concentration (males, all dates with a peak in Week 26), statistically significantly increased TSH (Week 26 and 105, females), all compared to controls.

Enzyme activities in the liver
- 50 ppm: statistically significantly increased ECOD (males), ALD (males) and EH (females), all compared to controls.
- 500 ppm: statistically significantly increased ECOD (males/females), ALD (males/females), EH (females), GST (males/females) and UDP-GT (males/females), all compared to controls.
- 1000 ppm: statistically significantly increased ECOD (males, up to a factor of 4/females, up to a factor of 3.4), ALD (males, up to a factor of 2/females, up to a factor of 2.5), EH (males, up to a factor of 2/females, up to a factor of 3.3), GST (males, up to a factor of 1.5/females, up to a factor of 1.6) and UDP-GT (males, up to a factor of 1.9/females, up to a factor of 2.5), all compared to controls.

In case of the 25 ppm dose level, ECOD and ALD were induced in males and EH in females. To test the validity of these findings, additional liver samples were analyzed (Attachment 6, Table 6 (b)) that could not support these data. Therefore, the increased ECOD, ALD and EH activity in males and females at 25 ppm compared to controls is seen as incidental. At the higher dose levels, the determination of enzymes in liver homogenates revealed an induction of all measured enzymes (except EROD) in male and female rats.

Summarized data can be found in Attachment 5 (clinical chemistry), Attachment 6 (thyroid parameters) and Attachment 7 (liver enzyme activities) in the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No differences between control and treatment groups were observed for blood, bilirubin, glucose, protein, urobilinogen, ketone, pH and sediments. Several quantitative parameters (density, K, Ca) were statistically significantly different to control values at different time points, but no dose- or time-dependency could be established and/or the difference was not of biological significance.

Summarized data can be found in Attachment 8 in the attached background material.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Interim sacrifice:
- 25, 50 and 500 ppm: There were no effects on organ weights observed up to and including 500 ppm in males and females.
- 1000 ppm: significantly increased relative liver weights (+16%, females) compared to controls. There was also a trend observed in males (+9%) without reaching statistical significance.

Terminal sacrifice:
- 25, 50 ppm and 500 ppm: There were no effects on organ weights observed up to and including 500 ppm in males and females.
- 1000 ppm: increased absolute (males) and relative (males/females) liver weights compared to controls. Relative liver weights were increased by 31% in males and by 16% in females.

All other differences were not considered of biological significance as they did not show dose-dependency.

Summarized data can be found in Attachment 9 (interim sacrifice) and 10 (terminal sacrifice) in the attached background material.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Interim sacrifice: No differences were found between control and treatment groups.

Terminal sacrifice:
- 25 and 50 ppm: There were no effects observed up to and including 50 ppm in males and females.
- 500 ppm: increased incidence of cysts in the liver (females), thyroids showed enlargements and nodules (males, females), decreased incidence of nodules in the pituitary gland (females), slightly higher incidence of turbidity in the eye (females), all compared to controls.
- 1000 ppm: increased incidence of area / s (males) and cysts (females) in the liver, thyroids showed enlargements and nodules (males, females), increased incidence of nodules in the uterus, decreased incidence of nodules in the skin and in the pituitary gland (females), slightly higher incidence of turbidity in the eye (females), all compared to controls.

In an amendment to the study report the additional pathological and histopathological examination of the uterus in females of all dose groups from the interim sacrifice (previously only control, high dose group and gross abnormalities) was provided. There was no evidence of any gross finding in the uterus related to treatment with the test compound.

Summarized data can be found in Attachment 11 (terminal sacrifice) in the attached background material.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim sacrifice:
- 25 and 50 ppm: There were no effects on organ weights observed up to and including 50 ppm in males and females.
- 500 ppm: hepatocellular hypertrophy (5/10 males and 5/10 females), focal fat infiltration (5/10 males), hypertrophy in the thyroid (7/10 males), colloidal clumping in the thyroid (10/10 males)
- 1000 ppm: hepatocellular hypertrophy (9/10 males, 8/10 females), focal fat infiltration (7/10 males), hypertrophy in the thyroid (8/10 males, 4/10 females), colloidal clumping in the thyroid (10/10 males, 8/10 females)

Summarized data can be found in Attachment 12 in the attached background material.

Final necropsy:
- 50 ppm:
Eyes: significant increase in retinal atrophy compared to controls (females).
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males), increased mixed eosinophilic-clear cell foci in the liver (males), all compared to controls.
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males) compared to controls.
- 500 ppm:
Neurology: slightly increased radiculoneuropathy (females), sciatic nerve degeneration (males, females), all compared to controls.
Skeletal muscle: increased atrophy in skeletal muscles (females) compared to controls.
Eyes: significant increase in retinal atrophy (females), increased degeneration of lens fibers (cataract) in females, all compared to controls.
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males, females), increased mixed eosinophilic-clear cell foci in the liver (males), slightly increased hepatic focal necrosis and biliary cysts (females), decreased incidence of basophilic foci (males, females), reduced clear cell foci (females), all compared to controls.
Brain: increased incidence of cholesterol clefts in the space between the anterior pituitary and the pars intermedia (males).
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males, females), thyroid colloid alteration and pigment in the follicular epithelium (males, females), all compared to controls.
Female reproductive tract: higher incidences of ovarian cysts (females) compared to controls.
- 1000 ppm:
Neurology: increased cholesterol clefts in the nerve roots of the spinal cord (females), significantly increased radiculoneuropathy (females), sciatic nerve degeneration (males, females), all compared to controls.
Skeletal muscle: increased atrophy, degeneration and mononuclear infiltrates in skeletal muscles (females) compared to controls.
Eyes: significant increase in retinal atrophy (females), increased degeneration of lens fibers (cataract) in females, compared to controls.
Liver: hepatocellular cytoplasmic change (eosinophilic cytoplasm with basophilic strands) and predominantly centrilobular hepatocellular hypertrophy (males, females), increased incidences of hepatocellular vacuolation (males), slightly increased hepatic focal necrosis and biliary cysts (females), increased mixed eosinophilic-clear cell foci in the liver (males, females), decreased incidence of basophilic foci (males), reduced clear cell foci (females), all compared to controls.
Mesenteric: increased incidence of sinus histiocytosis in the mesenteric lymph node (females) compared to control.
Brain: increased incidence of cholesterol clefts in the space between the anterior pituitary and the pars intermedia (males), all compared to controls.
Thyroid: increased number of animals with hypertrophy of the follicular epithelium of the thyroid (males, females), thyroid colloid alteration and pigment in the follicular epithelium (males, females), all compared to controls.
Female reproductive tract: higher incidences of ovarian cysts (females), higher incidence of lacteal cysts and galactocele in mammary glands (females), all compared to controls.

The additional evaluation of the uterus in the Amendment I showed that glandular hyperplasia of the uterine mucosa was found more frequently and to higher degrees in treated females (500 and 1000 ppm) compared to controls. However, it is not clear whether this finding is related to treatment or to other findings in the uterus like endometritis or stromal polyps which are frequently found in females of this strain and age and were also found in this study. Namely, endometritis was seen in one control and one female of the 500 ppm group, a stromal polyp occurred in an animal treated with 50 ppm and one female receiving 500 ppm had endometritis and a stromal polyp. Therefore, this finding is ambiguous.

As stated in the expert statement addressing target-organ findings after repeated exposure (M-282359-01-1), sciatic nerve degeneration is a common finding in aging rats and the muscle degeneration, which appears also spontaneously in untreated rats of high age, are most probably secondary to the changes in the nerve roots and in the sciatic nerve. Historical control data for sciatic nerve degeneration can be found in Attachment 18. Moreover, none of the observations was found at interim necropsy and other studies using even higher doses could not reproduce neurotoxicity. Therefore, a primary neurotoxic effect is unlikely.

The eye finding retinal atrophy is frequently observed as a spontaneous lesion in rats from control groups. In addition, the incidences are varying throughout the control groups. This can be seen from the table of incidences of retinal atrophy in control animals of chronic rat studies conducted at the testing laboratory in a similar time period (50 males and 50 females per group), which can be found in Attachment 18. Comparison of the incidences in the present study with the historical control data reveals that for retinal atrophy with control incidences of 3 - 21 per 50 animals in male rats and 12 - 29 per 50 animals in female rats, only the incidence at the highest dose exceeds the historical range slightly.

These non-neoplastic changes were considered as treatment-related. The hepatocellular hypertrophy is a known adaptive response giving evidence of liver enzyme induction. Summarized data can be found in Attachment 13 and 14 (additional uterus investigations) in the attached background material.

Several other obvious incidence differences, especially between high dose groups of interim and final sacrifices and controls may be partly related to reduced body weights/food consumption. These were reduced incidence of periarteritis in different organs and chronic progressive nephropathy in females, reduced incidence of medullary hyperplasia in the adrenals of males or reduced incidence of inflammatory infiltrates in the extraorbital lacrimal glands of females). They were considered secondary effects and not relevant for toxicological evaluation.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim necropsy: There were no stand-alone findings for neoplasia, but at 1000 ppm in males, one follicular cell adenoma in the thyroid was found, which is in line with the findings at terminal sacrifice.

Summarized data can be found in Attachment 15 in the attached background material.

Terminal sacrifice
- 25 and 50 ppm: There were no differences observed regarding neoplastic findings up to and including 50 ppm in males and females.
- 500 ppm: increased incidence of follicular cell adenoma of the thyroids (males), increased incidence of uterine adenocarcinoma (females) compared to controls.
- 1000 ppm: increased incidence of follicular cell adenoma of the thyroids (males, slightly elevated also in females), increased incidence of uterine adenocarcinoma compared to controls.

Other neoplasmas were decreased in treatment groups. Benign medullary tumors (phaeochromocytomas) of the adrenal glands occurred at lower incidence in males at 500 and 1000 ppm compared to controls. In females, incidence of fibroadenomas of the mammary gland and C-cell adenomas of the thyroid gland was reduced (1000 ppm) compared to controls.

Summarized results can be found in Attachment 16 in the attached background material.

Number of animals with tumors
- 1000 ppm: There is a slight increase in the number of animals with neoplasms, those with more than one neoplasm, and the number of primary (benign) neoplasms in 1000 ppm males, which can easily be explained by the increased thyroid follicular cell adenoma incidence. In females there is an increase in the number of animals with metastasising malignant tumors, which is caused by the higher uterine adenocarcinoma incidence.

Summarized results can be found in Attachment 17 in the attached background material.
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other:
Remarks on result:
other: corresponding to 1.2 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other:
Remarks on result:
other: corresponding to 3.3 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: corresponding to 2.5 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
500 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
Remarks on result:
other: corresponding to 33.5 mg/kg bw/day
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Historical control data from 1995 and 1998 can be found in the attached background material (Attachment 16 and 17).

Conclusions:
In this study, the long term toxicity and carcinogenic effects of the test substance were investigated in rats. The study was conducted according to OECD guideline 453 with deviations and under GLP conditions. The deviations were missing examinations, namely blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology was performed and the feed was not analyzed for phytoestrogens. Nevertheless, the study meets the criteria of a key study and is valid.

After long-term administration of the test substance via the diet, hepatic enzyme induction, namely of 7-ethoxycoumarin-deethylase (ECOD), aldrin-epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GS-T) and UDP-Glucuronyl-transferase (UDP-GT), was shown to be a toxicologically relevant effect in rats. Most enzyme activities were statistically significantly increased in males and females at 500 ppm and higher doses. In addition, the hepatic markers aspartate-aminotransferase (ASAT) and alkaline phosphatase (APh) were increased in males (Week 53). Macroscopically and histopathologically, the liver was altered, emphasizing the hepatic effect of the test substance. Secondary effects were thyroidal alterations, both non-neoplastic and neoplastic, probably due to increased T4 turnover followed by increased TSH release and thyroidal hyperstimulation. Tumors were also found in the uterus along with histopathological and macropathological observations. These findings may be considered to be secondary to the liver enzyme induction. Changes in liver enzymes as well as liver pathology in general were more pronounced in males, as were thyroidal tumors. In females the test item induced more pronounced effects on the nervous system and skeletal muscles. However, as discussed above, these were only relevant at the higher dose levels and can be seen as a secondary effect to changes in the nerve roots. Oculotoxic effects were mainly observed in females at the two highest dose levels (500 and 1000 ppm) and after life-long treatment only. The test substance may not directly induce cancer, but formation of cancer was observed at high doses of the test substance (500 and 1000 ppm) probably as secondary effect to hepatic enzyme induction. The NOAEL for general systemic toxicity in males was set at 25 ppm and for females at 50 ppm (corresponding to 1.2 mg/kg bw/day and 3.3 mg/kg bw/day, respectively). In the rat, the liver was identified as the main target organ after long-term dietary administration. But although there were changes in some clinical chemistry parameters (liver enzymes and thyroid hormones) at dose levels potentially relevant for classification according to CLP Regulation, these changes are considered indicative of increased liver and thyroid activity as the result of adaptive changes. Such adaptive responses are not considered adverse changes indicative of specific target organ toxicity relevant for CLP classification. Tumors were seen in males and females at 500 ppm (thyroid and uterine, respectively, corresponding to 25.2 and 33.5 mg/kg bw/day).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.2 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The quality of the database is good, several key studies are available in different species to elucidate the repeated dose toxicity of the test substance.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Apr - 4 Jun 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Blood was taken from non-fasted animals for glucose determination, no BALF analysis or determination of lung burden was conducted, humidity in the exposure chamber was lower than recommended
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used for toxicology studies, recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 243 - 246 g (group means, males), 166 - 171 g (group means, females)
- Fasting period before study: no
- Housing: individually in in conventional Makrolon® Type II cages with low-dust wood granulate (type S 8/15, Ssniff, Soest/Westfalen, Germany)
- Diet: standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 10 days

DETAILS OF FOOD AND WATER QUALITY: food and water were checked for contaminations regularly

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 May To: 4 June 1997
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.9 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure retainers, designed that the rats tail remained outside to prevent hyperthermia.
- Method of holding animals in test chamber: Plexiglas exposure retainers are designed to hold the rat in place for exposure.
- System of generating particulates/aerosols: The test substance was aerosolized using a Wright-Dust-Feeder (BGI Inc., Waltham, MA, USA). For powder dispersion, conditioned compressed air (30 liters of air/min) was used. A cyclone prevented larger particles (>10 µm) from entering into the exposure chamber.
- Temperature, humidity in air chamber: 21 - 22 °C, 11 - 18%
- Inlet Air flow: 30 L/min
- Air change rate: 30 L/min x 60 min/(3.8 L) = 474 L
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).
- Treatment of exhaust air: The exhaust air was purified via cotton-wool and HEPA filters which were disposed.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: cellulose-acetate filter, Sartorius, Göttingen, Germany; balance: MettlerAE 100). Samples for particle size analysis were also taken from the breathing-zone.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As mentioned previously, the concentrations were determined by gravimetric analysis; The number of samples taken was sufficient to characterize the test atmosphere and was adjusted to accommodate sampling duration and/or the need to confirm specific concentration values. It was intended to collect at least three samples/exposure, after the equilibrium concentration was reached. The analytical concentrations were reported as mg of test item/m³ air.
The amount of particles below 3 μm was 54.5 (2 mg/m³), 51.6 (20 mg/m³) and 53.1% (200 mg/m³).
Duration of treatment / exposure:
28 days, 6 h/day
Frequency of treatment:
5 days per week
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 2.0 mg/m³
Dose / conc.:
20 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 18.2 mg/m³
Dose / conc.:
200 mg/m³ air (nominal)
Remarks:
nominal concentration in air, had to be reduced to 100 mg/m³ in the second week, gravimetric concentration in air was 143.4 mg/m³ (mean over entire testing period)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were selected based on the results of an acute inhalation study and a one-week pilot study, both conducted on rats. In the acute toxicity study, rats were exposed to the test substance at concentrations of 80, 481, 1523, and 2535 mg/m³ air. The NOAEL was found to be 80 mg/m³ air for males and females and the LC50 > 2535 mg/m³ for males and approximately 1223 mg/m³ in females.
In the pilot study, rats were exposed to the test substance at 1.97, 19 or 205 mg/m³ air. Only minor influences on the respiratory tract were found up to the highest concentration tested. At 205 mg/m³ air, liver weight was increased transiently and induced hepatic enzyme induction was found. At 19 mg/m³, grip strength was increased transiently and an increased incidence of darken spleens was noticed in the females of this group. All findings were reversible within 2 weeks. 19 mg/m³ was therefore seen as the lowest effect level. Based on these results, the concentrations for the present study were set at approximately 2, 20 and 200 mg/m³ air. 2 mg/m³ was expected to be non-toxic while 20 and 200 mg/m³ air should induce toxic effects. However, during the course of the study, the highest concentration of 200 mg/m3 needed to be reduced to 100 mg/m3 air to prevent undue distress of animals or even mortality.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and holidays)
- Cage side observations: changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to first exposure and towards end of the exposure period
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal sacrifice by cardiac puncture or, for glucose determination, blood was sampled next to last study week (after urine collection) from the caudal vein
- Anesthetic used for blood collection: Yes (pentobarbital), not for blood withdrawal for glucose determination
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, thrombocyte count, reticulocytes, Heinz' bodies, leukocyte differential count (lymphocytes, granulocytes, segmented neutrophils, eosinophilic neutrophils, basophils, monocytes, plasma cells and miscellaneous abnormal cell types)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above (hematology)
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: aspartate aminotransferase, optimized (AST), alanine aminotransferase, optimized (ALT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), alkaline phosphatase (APh), y-glutamyltranspeptidase (GGT), albumin, blood glucose, urea, bilirubin, bile acids, creatinine, total protein, triglycerides, cholesterol, serum protein electrophoresis, clotting time (Hepato quick test), sodium, potassium, calcium, magnesium, phosphate, chloride

From liver samples, the following parameters were checked: N-DEM (N-Demethylase, Aminopyrin-N-demethylase), O-DEM (O-Demethylase, p-Nitroanisol-N-demethylase), cytochrome P450, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight during the next to last study week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Sediment composition, urine osmolality, specific density, pH, volume, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies
Protein (Q)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice daily (once on weekends and holidays)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No
Sacrifice and pathology:
Surviving animals were sacrificed one day after the last exposure to the test substance after cardiac exsanguination using sodium pentobarbital (i.p.) for euthanasia.

GROSS PATHOLOGY: Yes

Organ weight was determined for the following organs: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, thyroid (half), uterus

Relative organ to body and organ to brain weight was calculated.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenal glands, aorta, brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular and mesenteric), mammary gland, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary region), skin (muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroid gland with parathyroid glands, tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes wee also fixed if necessary.

- animals examined: for nasal cavity, larynx, trachea, lungs, lung associated lymph nodes,
heart, liver, kidneys, thyroid with parathyroid glands and esophagus all animals were examined, for all other organs control and high dose group were assessed, if differences occurred, the remaining groups were also checked.
- thickness of sections: 5 µm
embedding media: paraplast
- staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed livers were stained with Oil-Red-0 (ORO).
Other examinations:
Rectal temperature was assessed daily after cessation of exposure (within 30 min of cessation of exposure) using a digital thermometer.
Statistics:
All variables that are not dichotomous are described by sex, dosage group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, in most instances also minimum, maximum).

For normally distributed data with equal variances, an ANOVA followed by Dunnett's test is used. If heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. For non-parametric measurements, Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/m³, animals showed signs of morbidity (bradypnea, labored breathing pattern, reduced motility, piloerection, ungroomed hair-coat, atony, rales, salivation, mydriasis, miosis, tremor, vocalization). Therefore, the concentration was reduced to 100 mg/m³ in Week 2, the most severe signs (labored breathing, reduced mobility, ungroomed hair) decreased after having reduced the test concentration. It was concluded that most of the signs predominantly related to respiratory distress rather than
specific central nervous effects (mydriasis, miosis, tremor) which subsided completely after reduction of concentration.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: decreased body weight in the first week (this was reversible after reducing the concentration to 100 mg/m³)

Summarized data can be found in Attachment 2 in the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional findings were elevated MCHC in females of the low and middle dose group, increased neutrophils in males of the middle dose group, and decreased lymphocyte counts in the female high dose group. However, none of these were dose dependent and were therefore considered not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: statistically significant increase in alkaline phosphatase (females), phosphate (males), glucose (males, females), cholesterol, bile acids (females) and calcium (females), all compared to controls

APh and phosphate were also increased in the other sex but not statistically significant. Females were mildly more susceptible than males.

Liver tissues
- 20 mg/m³: statistically significantly increased N-DEM (males) compared to controls
- 200 mg/m³: statistically significantly increased N-DEM (males, females), O-DEM (males, females), P450 (males, females), all compared to controls

In females at 2 mg/m³, N-Dem was decreased statistically significantly compared to controls. This was considered incidental.

Summarized results can be found in Attachment 3 of the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: statistically significantly increased absolute (females +16.8%) and relative to body (males +12.2%, females +13.8%) liver weight, female livers were also statistically significantly increased relative to brain weight (+16.1%), all compared to controls

In males of the 2 and 200 mg/m³ groups the lung weights were statistically significantly increased, however, there was no evidence of any consistent concentration-dependence of effect. Thyroid weights were mildly increased in the middle and high dose group (absolute weight was increased 54.8% at 20 mg/m³ and 37.1% at 200 mg/m³) but this was not statistically significant.

Summarized results can be found in Attachment 4 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences between control and control groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
The assessment of reflexes did not reveal any differences between the groups. The only sign observed was that on day 2, some rats of the 200 mg/m³ group experienced a decreased tonus and light reflex (reversible).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 20 mg/m³: slight hypertrophy of hepatocytes (males)
- 200 mg/m³: slight hypertrophy of hepatocytes (males, females), hypertrophy of the thyroidal follicular epithelium (males)

Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal temperature
- 200 mg/m³: decreased body temperature (34.1 °C for males and 34.5/35.3°C for females in Week 1).

Summarized data can be found in Attachment 5 in the attached background material.
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
18.2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to the nominal concentration of 20 mg/m³
Key result
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³
Key result
Dose descriptor:
NOAEC
Remarks:
respiratory system
Effect level:
>= 143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on the respiratory system was found up to the highest concentration tested.
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
18.2 mg/m³ air
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 (dated 1981) and GLP conditions. Male and female rats were exposed to the test item as aerosol at measured concentrations of 2, 18.2, and 143.4 mg/m³ air, under dynamic directed-flow nose-only exposure conditions. The aerosol was highly respirable to rats, with an average mass median aerodynamic diameter (MMAD) of 2.9 µm. Signs of hepatotoxicity were noticed at the highest tested concentration (liver enzyme induction, minimal to slight hepatocellular hypertrophy); furthermore, slight thyroid follicular cell hypertrophy also was noticed. Accordingly, the LOAEC was set at 143.4 mg/m³ whereas the NOAEC was 18.2 mg/m³.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
18.2 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
The quality of the database is sufficient to draw the conclusion described.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Apr - 4 Jun 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Blood was taken from non-fasted animals for glucose determination, no BALF analysis or determination of lung burden was conducted, humidity in the exposure chamber was lower than recommended
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used for toxicology studies, recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 243 - 246 g (group means, males), 166 - 171 g (group means, females)
- Fasting period before study: no
- Housing: individually in in conventional Makrolon® Type II cages with low-dust wood granulate (type S 8/15, Ssniff, Soest/Westfalen, Germany)
- Diet: standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 10 days

DETAILS OF FOOD AND WATER QUALITY: food and water were checked for contaminations regularly

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 May To: 4 June 1997
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.9 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure retainers, designed that the rats tail remained outside to prevent hyperthermia.
- Method of holding animals in test chamber: Plexiglas exposure retainers are designed to hold the rat in place for exposure.
- System of generating particulates/aerosols: The test substance was aerosolized using a Wright-Dust-Feeder (BGI Inc., Waltham, MA, USA). For powder dispersion, conditioned compressed air (30 liters of air/min) was used. A cyclone prevented larger particles (>10 µm) from entering into the exposure chamber.
- Temperature, humidity in air chamber: 21 - 22 °C, 11 - 18%
- Inlet Air flow: 30 L/min
- Air change rate: 30 L/min x 60 min/(3.8 L) = 474 L
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).
- Treatment of exhaust air: The exhaust air was purified via cotton-wool and HEPA filters which were disposed.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: cellulose-acetate filter, Sartorius, Göttingen, Germany; balance: MettlerAE 100). Samples for particle size analysis were also taken from the breathing-zone.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As mentioned previously, the concentrations were determined by gravimetric analysis; The number of samples taken was sufficient to characterize the test atmosphere and was adjusted to accommodate sampling duration and/or the need to confirm specific concentration values. It was intended to collect at least three samples/exposure, after the equilibrium concentration was reached. The analytical concentrations were reported as mg of test item/m³ air.
The amount of particles below 3 μm was 54.5 (2 mg/m³), 51.6 (20 mg/m³) and 53.1% (200 mg/m³).
Duration of treatment / exposure:
28 days, 6 h/day
Frequency of treatment:
5 days per week
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 2.0 mg/m³
Dose / conc.:
20 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 18.2 mg/m³
Dose / conc.:
200 mg/m³ air (nominal)
Remarks:
nominal concentration in air, had to be reduced to 100 mg/m³ in the second week, gravimetric concentration in air was 143.4 mg/m³ (mean over entire testing period)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were selected based on the results of an acute inhalation study and a one-week pilot study, both conducted on rats. In the acute toxicity study, rats were exposed to the test substance at concentrations of 80, 481, 1523, and 2535 mg/m³ air. The NOAEL was found to be 80 mg/m³ air for males and females and the LC50 > 2535 mg/m³ for males and approximately 1223 mg/m³ in females.
In the pilot study, rats were exposed to the test substance at 1.97, 19 or 205 mg/m³ air. Only minor influences on the respiratory tract were found up to the highest concentration tested. At 205 mg/m³ air, liver weight was increased transiently and induced hepatic enzyme induction was found. At 19 mg/m³, grip strength was increased transiently and an increased incidence of darken spleens was noticed in the females of this group. All findings were reversible within 2 weeks. 19 mg/m³ was therefore seen as the lowest effect level. Based on these results, the concentrations for the present study were set at approximately 2, 20 and 200 mg/m³ air. 2 mg/m³ was expected to be non-toxic while 20 and 200 mg/m³ air should induce toxic effects. However, during the course of the study, the highest concentration of 200 mg/m3 needed to be reduced to 100 mg/m3 air to prevent undue distress of animals or even mortality.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and holidays)
- Cage side observations: changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to first exposure and towards end of the exposure period
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal sacrifice by cardiac puncture or, for glucose determination, blood was sampled next to last study week (after urine collection) from the caudal vein
- Anesthetic used for blood collection: Yes (pentobarbital), not for blood withdrawal for glucose determination
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, thrombocyte count, reticulocytes, Heinz' bodies, leukocyte differential count (lymphocytes, granulocytes, segmented neutrophils, eosinophilic neutrophils, basophils, monocytes, plasma cells and miscellaneous abnormal cell types)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above (hematology)
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: aspartate aminotransferase, optimized (AST), alanine aminotransferase, optimized (ALT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), alkaline phosphatase (APh), y-glutamyltranspeptidase (GGT), albumin, blood glucose, urea, bilirubin, bile acids, creatinine, total protein, triglycerides, cholesterol, serum protein electrophoresis, clotting time (Hepato quick test), sodium, potassium, calcium, magnesium, phosphate, chloride

From liver samples, the following parameters were checked: N-DEM (N-Demethylase, Aminopyrin-N-demethylase), O-DEM (O-Demethylase, p-Nitroanisol-N-demethylase), cytochrome P450, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight during the next to last study week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Sediment composition, urine osmolality, specific density, pH, volume, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies
Protein (Q)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice daily (once on weekends and holidays)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No
Sacrifice and pathology:
Surviving animals were sacrificed one day after the last exposure to the test substance after cardiac exsanguination using sodium pentobarbital (i.p.) for euthanasia.

GROSS PATHOLOGY: Yes

Organ weight was determined for the following organs: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, thyroid (half), uterus

Relative organ to body and organ to brain weight was calculated.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenal glands, aorta, brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular and mesenteric), mammary gland, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary region), skin (muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroid gland with parathyroid glands, tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes wee also fixed if necessary.

- animals examined: for nasal cavity, larynx, trachea, lungs, lung associated lymph nodes,
heart, liver, kidneys, thyroid with parathyroid glands and esophagus all animals were examined, for all other organs control and high dose group were assessed, if differences occurred, the remaining groups were also checked.
- thickness of sections: 5 µm
embedding media: paraplast
- staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed livers were stained with Oil-Red-0 (ORO).
Other examinations:
Rectal temperature was assessed daily after cessation of exposure (within 30 min of cessation of exposure) using a digital thermometer.
Statistics:
All variables that are not dichotomous are described by sex, dosage group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, in most instances also minimum, maximum).

For normally distributed data with equal variances, an ANOVA followed by Dunnett's test is used. If heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. For non-parametric measurements, Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/m³, animals showed signs of morbidity (bradypnea, labored breathing pattern, reduced motility, piloerection, ungroomed hair-coat, atony, rales, salivation, mydriasis, miosis, tremor, vocalization). Therefore, the concentration was reduced to 100 mg/m³ in Week 2, the most severe signs (labored breathing, reduced mobility, ungroomed hair) decreased after having reduced the test concentration. It was concluded that most of the signs predominantly related to respiratory distress rather than
specific central nervous effects (mydriasis, miosis, tremor) which subsided completely after reduction of concentration.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: decreased body weight in the first week (this was reversible after reducing the concentration to 100 mg/m³)

Summarized data can be found in Attachment 2 in the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No differences between control and treatment groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional findings were elevated MCHC in females of the low and middle dose group, increased neutrophils in males of the middle dose group, and decreased lymphocyte counts in the female high dose group. However, none of these were dose dependent and were therefore considered not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: statistically significant increase in alkaline phosphatase (females), phosphate (males), glucose (males, females), cholesterol, bile acids (females) and calcium (females), all compared to controls

APh and phosphate were also increased in the other sex but not statistically significant. Females were mildly more susceptible than males.

Liver tissues
- 20 mg/m³: statistically significantly increased N-DEM (males) compared to controls
- 200 mg/m³: statistically significantly increased N-DEM (males, females), O-DEM (males, females), P450 (males, females), all compared to controls

In females at 2 mg/m³, N-Dem was decreased statistically significantly compared to controls. This was considered incidental.

Summarized results can be found in Attachment 3 of the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: statistically significantly increased absolute (females +16.8%) and relative to body (males +12.2%, females +13.8%) liver weight, female livers were also statistically significantly increased relative to brain weight (+16.1%), all compared to controls

In males of the 2 and 200 mg/m³ groups the lung weights were statistically significantly increased, however, there was no evidence of any consistent concentration-dependence of effect. Thyroid weights were mildly increased in the middle and high dose group (absolute weight was increased 54.8% at 20 mg/m³ and 37.1% at 200 mg/m³) but this was not statistically significant.

Summarized results can be found in Attachment 4 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences between control and control groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
The assessment of reflexes did not reveal any differences between the groups. The only sign observed was that on day 2, some rats of the 200 mg/m³ group experienced a decreased tonus and light reflex (reversible).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 20 mg/m³: slight hypertrophy of hepatocytes (males)
- 200 mg/m³: slight hypertrophy of hepatocytes (males, females), hypertrophy of the thyroidal follicular epithelium (males)

Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal temperature
- 200 mg/m³: decreased body temperature (34.1 °C for males and 34.5/35.3°C for females in Week 1).

Summarized data can be found in Attachment 5 in the attached background material.
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
18.2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to the nominal concentration of 20 mg/m³
Key result
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³
Key result
Dose descriptor:
NOAEC
Remarks:
respiratory system
Effect level:
>= 143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on the respiratory system was found up to the highest concentration tested.
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
18.2 mg/m³ air
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 (dated 1981) and GLP conditions. Male and female rats were exposed to the test item as aerosol at measured concentrations of 2, 18.2, and 143.4 mg/m³ air, under dynamic directed-flow nose-only exposure conditions. The aerosol was highly respirable to rats, with an average mass median aerodynamic diameter (MMAD) of 2.9 µm. Signs of hepatotoxicity were noticed at the highest tested concentration (liver enzyme induction, minimal to slight hepatocellular hypertrophy); furthermore, slight thyroid follicular cell hypertrophy also was noticed. Accordingly, the LOAEC was set at 143.4 mg/m³ whereas the NOAEC was 18.2 mg/m³.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
143.4 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is sufficient to draw the conclusion described.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep - 2 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used specied for toxicological studies and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks (males), 14 weeks or older (females)
- Weight at study initiation: 222 - 256 g (males), 208 - 235 g (females)
- Fasting period before study: not applicable
- Housing: individually in polycarbonate cages type IIA with low-dust wood shavings, during acclimation in groups of 3 or 5 in polycarbonate cages type III
- Diet: Altromin 1324 Diet for Rats and Mice (Altromin GmbH and Co. KG in Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly analyzed for contaminations

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2.0
- Humidity (%): approx. 55 ± 5
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
only moistened by tap water
Details on exposure:
TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: over 10%
- Type of wrap if used: gauze layer ("Hansapor steril" patch), secured in place using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm), at weekends, a ,"Fermoflex®" dressing was used additionally
- Time intervals for shavings or clippings: one day before first day of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing: with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the amount was weighed out onto the gauze layer ("Hansapor steril" patch)
- Constant volume or concentration used: no
- For solids, paste formed: no, the powder was only moistened with tap water


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical verification was not performed because the substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:
4 weeks treatment, 2 weeks recovery
Frequency of treatment:
First three weeks: 5 days/week, 4th week: 7 days/week
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.4-6.0 mg/cm²
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.1-5.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 6.7-9.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the recovery group, corresponding to 7.0-9.4 mg/cm²
No. of animals per sex per dose:
5 (main and recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were based on a previously conducted two-week dose range-finding study in male and female rats. At 1000 mg/kg bw/day, slightly lower feed consumption in Week 1 (males and females), decreased lymphocyte counts and increased counts of segmented neutrophils (females), increased cholesterol (males) and decreased triglyceride levels (females), increased liver weight (males) and lower thymus weights (males and females), and spleens of dark-red color (females) were observed. On the application area neither erythema nor edema of the skin were observed.
- Rationale for selecting satellite groups: reversibility of the observations should be assessed
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, (once daily at weekends and on public holidays)
- Cage side observations: Mortality, moribund state, body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes (scoring according to Draize, including swelling)
- Time schedule for examinations: daily before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 0 before before the first application

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23/24 (main groups) and 41/42 (recovery groups) for glucose, on day of necropsy (Day 28 for main groups, Day 43 and 44 for recovery groups) for all other parameters
- Anesthetic used for blood collection: Yes (diethyl ether anesthesia, for all parameters except glucose) / No (glucose)
- Animals fasted: Yes (for glucose determination, blood taken from the distal vessels of the tail prior to necropsy) /No (everything else, blood taken by heart puncture at necropsy)
- How many animals: all animals
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, hepato quick, leucocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume erythrocytes (MCV), thrombocytes (THROMBO) and differential blood count (eosinophils, lymphocytes, monocytes, normal RBC, segmented neutrophils).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: No
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALAT), albumin, alkaline phosphatase (APh), aspartate aminotransferase (ASAT), bilirubin total, cholesterol, creatinine, calcium, chloride, gamma-glutamyltransferase (GGT), glutamate dehydrogenase (GLDH), potassium, sodium, inorganic phosphorus, protein, triglycerides, urea and glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination under deep ether anesthesia.

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes and thymus.

HISTOPATHOLOGY: Yes
The following organs were immersion-fixed in Davidson's solution: Adrenal glands, brain (cerebrum, cerebellum, pons/medulla), heart, kidneys, liver, lung, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings.
One liver lobe and lungs (fixed by instillation of 4% aqueous formaldehyde solution) of all rats were immersion-fixed in 4% aqueous formaldehyde solution.

- embedding media: paraplast
- Thickness: 5 µm
- Staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed liver lobe were stained with Oil-Red-O (ORO)

Histopathological examination was limited to the following organs/groups:
Liver (HE), thyroid gland and necropsy findings: all animal groups (including the recovery group)
Skin treated and untreated, liver (ORO): main groups (0, 100, 300, 1000 ppm, no recovery groups)
Adrenal glands, brain, heart, kidneys, lungs, spleen, testes, thymus: control and high dose group (main group only)
Statistics:
The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p < 0.05 and "++" for p < 0.01. In case of numbers of values too low to calculate test statistics this is indicated by "0".

The Dunnett test was used for body weight, body weight gain, feed consumption and organ
weight data (relative organ weights subsequent to logarithmic transformation).
If possible, an ANOVA was used followed by a Dunnett's test for parametric measurements. For non-parametric data, a Kruskal-Wallis test followed by adjusted U test was performed.

Macro- and micropathological data were processed with the PATHDATA program, version 3.6. B.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant difference between control and treatment group was observed.
Dermal irritation:
no effects observed
Description (incidence and severity):
The skin redness and skin thickness were comparable between control and treated animals.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference between control and treated animals was observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: transient decrease of feed intake in females in the first week compared to controls

This significant transient decrease could also be a sign of stress caused by the treatment rather than the test substance so this finding is ambiguous. As the effect is transient, it is not considered toxicologically relevant. The statistically significantly increased values for feed consumption in g/kg body weight and day that was recorded for males of the recovery group are considered incidental and a result of the slightly lower body weights of these animals, as the mean feed intakes per animal and day show no abnormalities.

Summarized data can be found in Attachment 1 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treatment groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences were observed in males of the main groups regarding cholesterol (100 and 300 mg/kg bw/day, -18.1 and -16.7%, respectively), creatinine (-13% for 100 mg/kg bw/day and -14.8% for 300 mg/kg bw/day), protein (-4.5% for 100 mg/kg bw/day and -6.3% for 300 mg/kg bw/day), albumin (-8.5% for 300 mg/kg bw/day) and glucose (-7.9% for 300 mg/kg bw/day) compared to controls. They are considered as incidental as they are not dose related and were observed only in one sex. At the end of the recovery period no toxicologically significant changes compared to controls were observed, the only statistically significant observations was slightly decreased creatine in males, considered incidental.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg bw/day: increased absolute (+12.7%/+13.5%) and relative (+17.2%/+11.9%) liver weights (males/females) in the main group. This finding was reversible in the recovery group, so that this observation can therefore be seen as part of an adaptive process rather than a toxicologically relevant effect.

Summarized data can be found in Attachment 2 of the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant differences between control and treatment groups were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 300 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (3/5 males) in the main group compared to controls. At the end of recovery period these changes were still observed in males that received 1000 mg/kg bw/day (2/5).
- 1000 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (5/5 males, 3/5 females) compared to controls. At the end of the recovery period, this was still observed in males (2/5) but not in females. In the main group, hypertrophy of the follicular epithelium in thyroids (3/5 males, 2/5 females, all compared to controls) was observed. These findings were reversible for females but only partially in males, since hypertrophy of the follicular epithelium in thyroids occurred in 1/5 males of the recovery group.

The changes are considered to be toxicologically relevant.

Summarized results can be found in Attachment 3 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
dermal, local
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to this dose level.
Remarks on result:
other: Highest dose tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused liver and thyroid changes when applied dermally for four weeks in males and females. The changes in liver and thyroid were fully reversible for females but only partly reversible in males within the two-week recovery period. No dermal irritation was seen. Thus, 1000 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 100 and 300 mg/kg bw for males and females, respectively.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is sufficient to draw the conclusion described.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep - 2 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used specied for toxicological studies and recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks (males), 14 weeks or older (females)
- Weight at study initiation: 222 - 256 g (males), 208 - 235 g (females)
- Fasting period before study: not applicable
- Housing: individually in polycarbonate cages type IIA with low-dust wood shavings, during acclimation in groups of 3 or 5 in polycarbonate cages type III
- Diet: Altromin 1324 Diet for Rats and Mice (Altromin GmbH and Co. KG in Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly analyzed for contaminations

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2.0
- Humidity (%): approx. 55 ± 5
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
only moistened by tap water
Details on exposure:
TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: over 10%
- Type of wrap if used: gauze layer ("Hansapor steril" patch), secured in place using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm), at weekends, a ,"Fermoflex®" dressing was used additionally
- Time intervals for shavings or clippings: one day before first day of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing: with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the amount was weighed out onto the gauze layer ("Hansapor steril" patch)
- Constant volume or concentration used: no
- For solids, paste formed: no, the powder was only moistened with tap water


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical verification was not performed because the substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:
4 weeks treatment, 2 weeks recovery
Frequency of treatment:
First three weeks: 5 days/week, 4th week: 7 days/week
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.4-6.0 mg/cm²
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.1-5.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 6.7-9.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the recovery group, corresponding to 7.0-9.4 mg/cm²
No. of animals per sex per dose:
5 (main and recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were based on a previously conducted two-week dose range-finding study in male and female rats. At 1000 mg/kg bw/day, slightly lower feed consumption in Week 1 (males and females), decreased lymphocyte counts and increased counts of segmented neutrophils (females), increased cholesterol (males) and decreased triglyceride levels (females), increased liver weight (males) and lower thymus weights (males and females), and spleens of dark-red color (females) were observed. On the application area neither erythema nor edema of the skin were observed.
- Rationale for selecting satellite groups: reversibility of the observations should be assessed
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, (once daily at weekends and on public holidays)
- Cage side observations: Mortality, moribund state, body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes (scoring according to Draize, including swelling)
- Time schedule for examinations: daily before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 0 before before the first application

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23/24 (main groups) and 41/42 (recovery groups) for glucose, on day of necropsy (Day 28 for main groups, Day 43 and 44 for recovery groups) for all other parameters
- Anesthetic used for blood collection: Yes (diethyl ether anesthesia, for all parameters except glucose) / No (glucose)
- Animals fasted: Yes (for glucose determination, blood taken from the distal vessels of the tail prior to necropsy) /No (everything else, blood taken by heart puncture at necropsy)
- How many animals: all animals
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, hepato quick, leucocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume erythrocytes (MCV), thrombocytes (THROMBO) and differential blood count (eosinophils, lymphocytes, monocytes, normal RBC, segmented neutrophils).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: No
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALAT), albumin, alkaline phosphatase (APh), aspartate aminotransferase (ASAT), bilirubin total, cholesterol, creatinine, calcium, chloride, gamma-glutamyltransferase (GGT), glutamate dehydrogenase (GLDH), potassium, sodium, inorganic phosphorus, protein, triglycerides, urea and glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination under deep ether anesthesia.

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes and thymus.

HISTOPATHOLOGY: Yes
The following organs were immersion-fixed in Davidson's solution: Adrenal glands, brain (cerebrum, cerebellum, pons/medulla), heart, kidneys, liver, lung, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings.
One liver lobe and lungs (fixed by instillation of 4% aqueous formaldehyde solution) of all rats were immersion-fixed in 4% aqueous formaldehyde solution.

- embedding media: paraplast
- Thickness: 5 µm
- Staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed liver lobe were stained with Oil-Red-O (ORO)

Histopathological examination was limited to the following organs/groups:
Liver (HE), thyroid gland and necropsy findings: all animal groups (including the recovery group)
Skin treated and untreated, liver (ORO): main groups (0, 100, 300, 1000 ppm, no recovery groups)
Adrenal glands, brain, heart, kidneys, lungs, spleen, testes, thymus: control and high dose group (main group only)
Statistics:
The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p < 0.05 and "++" for p < 0.01. In case of numbers of values too low to calculate test statistics this is indicated by "0".

The Dunnett test was used for body weight, body weight gain, feed consumption and organ
weight data (relative organ weights subsequent to logarithmic transformation).
If possible, an ANOVA was used followed by a Dunnett's test for parametric measurements. For non-parametric data, a Kruskal-Wallis test followed by adjusted U test was performed.

Macro- and micropathological data were processed with the PATHDATA program, version 3.6. B.
Clinical signs:
no effects observed
Description (incidence and severity):
No significant difference between control and treatment group was observed.
Dermal irritation:
no effects observed
Description (incidence and severity):
The skin redness and skin thickness were comparable between control and treated animals.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference between control and treated animals was observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: transient decrease of feed intake in females in the first week compared to controls

This significant transient decrease could also be a sign of stress caused by the treatment rather than the test substance so this finding is ambiguous. As the effect is transient, it is not considered toxicologically relevant. The statistically significantly increased values for feed consumption in g/kg body weight and day that was recorded for males of the recovery group are considered incidental and a result of the slightly lower body weights of these animals, as the mean feed intakes per animal and day show no abnormalities.

Summarized data can be found in Attachment 1 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treatment groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences were observed in males of the main groups regarding cholesterol (100 and 300 mg/kg bw/day, -18.1 and -16.7%, respectively), creatinine (-13% for 100 mg/kg bw/day and -14.8% for 300 mg/kg bw/day), protein (-4.5% for 100 mg/kg bw/day and -6.3% for 300 mg/kg bw/day), albumin (-8.5% for 300 mg/kg bw/day) and glucose (-7.9% for 300 mg/kg bw/day) compared to controls. They are considered as incidental as they are not dose related and were observed only in one sex. At the end of the recovery period no toxicologically significant changes compared to controls were observed, the only statistically significant observations was slightly decreased creatine in males, considered incidental.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg bw/day: increased absolute (+12.7%/+13.5%) and relative (+17.2%/+11.9%) liver weights (males/females) in the main group. This finding was reversible in the recovery group, so that this observation can therefore be seen as part of an adaptive process rather than a toxicologically relevant effect.

Summarized data can be found in Attachment 2 of the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant differences between control and treatment groups were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 300 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (3/5 males) in the main group compared to controls. At the end of recovery period these changes were still observed in males that received 1000 mg/kg bw/day (2/5).
- 1000 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (5/5 males, 3/5 females) compared to controls. At the end of the recovery period, this was still observed in males (2/5) but not in females. In the main group, hypertrophy of the follicular epithelium in thyroids (3/5 males, 2/5 females, all compared to controls) was observed. These findings were reversible for females but only partially in males, since hypertrophy of the follicular epithelium in thyroids occurred in 1/5 males of the recovery group.

The changes are considered to be toxicologically relevant.

Summarized results can be found in Attachment 3 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
dermal, local
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to this dose level.
Remarks on result:
other: Highest dose tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused liver and thyroid changes when applied dermally for four weeks in males and females. The changes in liver and thyroid were fully reversible for females but only partly reversible in males within the two-week recovery period. No dermal irritation was seen. Thus, 1000 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 100 and 300 mg/kg bw for males and females, respectively.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
6.7 mg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
The quality of the database is sufficient to draw the conclusion described. The NOAEL represents the lowest nominal dose applied to the animals treated at 1000 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

The test substance induced hepatic enzyme induction, especially induction of UDP-GT. Activated UDP-GT leads to increased T4-turnover and thereby increased TSH release and hyperstimulation of the thyroid. It can also lead to general hormone imbalance. Since hepatic enzymes of rodents are more susceptible to induction by xenobiotics, human relevance is limited which is also shown by the different effects in rodents and non-rodents in the studies described below. For details, please refer to the information given in the "Additional information". 

Additional information

Oral repeated dose toxicity

 

Repeated dose toxicity in rats - oral

In rats, one subacute, one subchronic and one chronic toxicity study were conducted, all according to OECD guidelines and as key studies to assess the toxicity of the test material after repeated administration over various time frames. Additionally, one pilot study and one supporting study were performed over 14 and 21 days, respectively. In these, the mode of action was elucidated further, focusing on hepatic enzyme induction and thyroid alterations.

 

Pilot oral toxicity study in rats

In a pilot study, the test material was tested in rats both in an acute setting (single exposure) as well as for 14 continuous days (M-000703-01-4). The acute part is described and discussed in the respective part of this dossier. For the 14 days administration, 3 male and 3 female rats per group were administered the test substance via gavage. The doses were 5, 10, 20, 60 and 120 mg/kg bw/day and Cremophor® EL 2% in demineralized water (v/v) was used as the vehicle.

 

No mortality was observed but at 120 mg/kg bw/day one male showed decreased reactivity and females receiving 60 mg/kg bw/day excreted less feces. Body weight and feed consumption were reduced at 60 mg/kg bw/day and above, corresponding to reduced body weight in these groups. Leucocyte count was lower in animals treated with 120 mg/kg bw/day and clinical chemistry parameters were altered as follows: slightly increased Alkaline phosphatase (APh) activity (females, 60 mg/kg bw/day and above), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activity (both sexes, higher dose). In the two highest doses tested, increased cholesterol values were found. However, most of these values were within the historical control range and only individual data for alkaline phosphatase and cholesterol exceeded the historical control range. Liver enzyme induction was seen as increased cytochrome P-450 content and increased activity for N-demethylases and O-demethylases or Phase-I enzymes from 5 mg/kg bw/day in males and 60 mg/kg bw/day for females and phase II enzymes from 10 mg/kg bw/day for males and 60 mg/kg bw/day for females. At 60 and 120 mg/kg bw/day, liver weight was also increased and histopathological alterations were found. Regarding immunotoxicological examinations, a significantly lower cell count for the spleen was found for males (120 mg/kg bw/day) and females exhibited a decreased number of lymph nodes at 20 mg/kg bw/day and above. Macrophage populations in the spleen and liver nodes were well activated overall from 20 mg/kg/day. Cell proliferation investigations showed a distinct zonal increase in proliferation in the liver of female rats. A reduction in cell proliferation was determined in the kidneys of female rats.

 

This pilot study showed the test material had an influence on hepatic enzyme induction and several immunotoxic parameters. Moreover, cell proliferation was altered in kidney and liver and body weight and feed consumption were reduced at high doses. No significant effects on triiodothyronine (T3), thyroxine (T4) and thyroxine binding capacity (TBC) were noted and thyroid-stimulating hormone (TSH) was not investigated. These findings were used in subsequent studies.

 

Subacute oral toxicity studies in rats

In a three week repeated dose study, rats were administered the test substance orally via the diet (M-030427-03-1). Groups of 10 rats per sex and dose group received 25, 100, 400 or 1600 ppm of the test substance, corresponding to 2.6, 9.0, 36.9 and 145.1 mg/kg bw/day for males and 3.1, 12.3, 44.6 and 190.8 mg/kg bw/day for females. The stability and homogeneity of the test substance in the diet was assessed analytically and the test substance was proved to be stable in the feed for 8 days at room temperature. The study was conducted according to OECD Guideline 407 and under GLP but with several deviations since its aim was to investigate the mechanism of thyroidal changes after treatment with the test material. Therefore, sensory reactivity was not assessed and no hematology was performed. Regarding clinical chemistry, only T3, T4, TSH, UDP-glucuronyl transferase (UDP-GT) levels and activity and plasma hormone levels were assessed. Organ weights were recorded for liver, uterus and the left thyroidal lobe only and histopathology was performed only for the thyroid. Moreover, the study ran for three weeks instead of 28 days. Cage side observations were performed daily; body weight and feed consumption were assessed weekly. Blood was collected 2 days before treatment (Day -2) and on Day 2, 7 and 14 via the retroorbital plexus, on day 21 or 22, blood was collected by cardiac puncture.

 

Animals of the high dose group showed reduced body weight (up to -10.5% for males and -13.0% in females) and body weight gains (-14.9% in males and -43.5% in females) as compared to controls. Feed consumption was also reduced for both males and females of the high dose group in the first week of treatment. UDP-GT levels and activity were increased in males and females at 400 and 1600 ppm (up to +252% for males and +221% for females). In the high dose animals, the liver was enlarged (10/10 males and 9/10 females) and marked lobular patterns were observed for 7/10 females.

 

With regard to thyroid parameters, T3 and T4 were reduced in males and females of the high dose groups. T3 decrease was only statistically significant on day 2, but T4 decrease was observable throughout the study for males (relative decrease of -11.31 to -38.88%) and for females on Day 2, 7 and 22 (-21.8 to -41.6%). TSH levels were increased throughout the study in males and females, statistically significantly on Day 14 in males (+532.8%), and on Day 7 (+127%) and on Day 22 (+247.7%) in females. Increase in TSH levels was also seen in males of the 400 ppm group, but did not reach statistical significance. Histopathological evaluation of the thyroid showed minimal to slight hypertrophy of the follicular epithelium in males at 400 and 1600 ppm and for females at 1600 ppm.

The activation of UDP-GT in the liver can be seen as the primary effect of the test substance and the consequent changes in hormone level and thyroid histopathology as a secondary effect due to activation of the hypothalamic-pituitary-thyroidal (HPT) axis. Induction of UDP-GT leads to increased T4 glucuronide formation and excretion of T4, resulting in decreased T3 and T4 levels. TSH levels increase to compensate for decreased T4 and T3 levels and lead to a continuous activation of the thyroid. This hypothesis was supported by the findings of this study (higher UDP-GT levels, lower T3 and T4 levels, increased TSH levels and hypertrophy of the follicular epithelium of the thyroid). Therefore, the study revealed mechanistic effects of the test substance. A dietary dose of 100 ppm (equal to 9.0 mg/kg bw/day for males and 12.3 mg/kg bw/day for females) was determined as a clear no-observed-adverse-effect level.

 

In a supporting study (M-000785-02-1) 5 male and female rats were fed with the test substance for 14 days at dose levels of 25, 100, 500 and 2000 ppm. This corresponded to 2.5, 11.2, 49.2 and 187.5 mg/kg bw/day for males and 2.3, 9.8, 49.5 and 187.2 mg/kg bw/day for females. Body weight and feed consumption were recorded as well as clinical signs. Clinical chemistry was performed with blood withdrawn on Day 7 and at necropsy and liver enzymes relevant for xenobiotic metabolism were assessed using liver tissues collected at necropsy. At necropsy, all animals were examined by gross pathology, the liver and the left thyroidal lobe were weighed and liver and thyroid tissues were evaluated histopathologically. The study was conducted according to OECD guideline 407 but with several deviations. However, the study is considered valid and reliable with acceptable restrictions.

The study supports the suggestions and findings of the three-week study. TSH, gamma-glutamyltransferase (GGT) and enzymes associated with cytochrome P450 enzymes were increased in the high-dose group. Moreover, higher cholesterol levels were found in males at 100 ppm and higher doses and in females of the high dose group. In the liver, macroscopic and gross pathological alterations were observed (mainly in the high dose groups of males and females, even though at least 1/5 females in every treatment group had distinct liver lobulation). Histopathologically, the liver was affected from 500 ppm onward in males and females (hypertrophy of centrolobular hepatocytes, combined with delicately structured cytoplasm). Males of the high dose group also had lipid storage in the liver and hypertrophy of the follicular epithelium of the thyroid glands. It can therefore be concluded that oral administration of the test material over 14 days in rats induced hepatic enzyme activity, which resulted in macroscopic and histopathological changes in the liver. The treatment also had an influence on the thyroid system. Since UDP-GT was induced at 500 and 1000 ppm, the influence on the TSH level can be seen as a consequence of increased T4 turnover in the liver, which is in line with the previous study. The NOAEL for liver enzyme induction was 100 ppm (corresponding to 11.2 mg/kg bw/day for males and 9.8 mg/kg bw/day for females). The study was conducted according to OECD guideline 407 with deviations (length of study, clinical chemistry and haematology parameters tested, limited organ and tissue collection) and under GLP.

 

Subchronic oral toxicity study in rats

A subchronic study was conducted in Wistar rats for 13 weeks, including recovery groups that were observed for 5 additional weeks after the end of treatment (M-000863-01-1, 1997). All groups consisted of 10 male and 10 female rats. The study was conducted according to OECD guideline 408 and under GLP conditions. However, there were some deviations from the guideline. For example, no sensory reactivity was assessed and lipid analysis in blood was only done for cholesterol, various organs were not weighed at necropsy and no estrus cycle was determined. Nevertheless, the study was considered valid and reliable.

Rats received either the unchanged feed or feed mixed with the test substance at concentrations of 25, 100, 400 or 1600 ppm. Recovery groups received either the control feed or the high dose. The actual ingested doses were 1.9, 7.3, 28.6 and 123.1 mg/kg bw/day for males and 2.0, 7.6, 35.6 and 160.6 mg/kg bw/day. Doses were based on a previously conducted subacute study with the test substance in which doses of 0, 5, 20, 60 and 120 mg/kg bw/day were tested. All test material treatments resulted in increased liver enzyme activity. From 20 mg/kg bw/day onward, water consumption was increased, cell counts in lymph nodes were reduced, and an increase in macrophage activation was observed. 60 mg/kg bw/day and higher doses led to decreased body weight, feed consumption and leucocyte counts. In the high dose group, animals showed decreased reactivity, slight effects in the liver were found, increased concentrations of anorganic phosphate, reduced cell counts in spleen and increased cell proliferation in liver tissue were observed additionally. Based on these results, 25, 100, 400 and 1600 ppm were selected as dose levels. The post-exposure period was set to 5 weeks to see whether alterations were reversible.

Dietary administration of the test substance had no toxicologically relevant effects on survival rate, state of health, or general behavior of the animals. Body weight and body weight gain were reduced in both sexes at 1600 ppm throughout the study until termination (-14 to -16%). This effect was not reversible during the recovery period but the difference diminished. Feed consumption was unaltered by treatment but males of the 1600 ppm group consumed more water. In females of the high dose, water consumption was higher in g per animal and per kg bw but total and daily water consumption did not change, so that the finding was considered to be of no toxicological relevance. Ophthalmological examination and histopathological investigation of the eyes did not reveal any toxic effects on the eye. Regarding urinalysis, the only finding was increased urine sodium in males receiving 1600 ppm of the test substance. However, the results of macroscopic and histopathological examinations, organ weights determination and examination of cell proliferation provided no indication of kidney damage at doses up to 1600 ppm.

In doses up to and including 100 ppm no influence on the liver was found. At 400 ppm and higher doses, histopathological changes were seen as moderate hepatocellular hypertrophy associated with a fine granular to vesicular structure in the cytoplasm in male and female rats. At the end of the recovery period, the finding was less pronounced but still observable in males but a cytoplasmatic change was no longer found. Absolute and relative liver weights were increased in males and females at 1600 ppm. Increased liver weight was already visible in males treated with 400 ppm for 13 weeks, however this finding was only statistically significant compared to controls relative to body weight. Absolute liver weights were slightly increased but without reaching statistical significance. Therefore, the altered weight of the livers in this dose group is not clearly related to the test substance administration. Changes in liver metabolism were also evident on the molecular level. Induction of hepatic enzymes of the xenobiotic metabolism (Phase I and II) as well as an increase in triglycerides were observed in males and females at 400 ppm and higher doses. Especially UDP-GT was increased 5.28 times in males and 4.59 times in females of the high dose group. The changes in liver enzyme activities and in liver weight were shown to be completely reversible during the recovery period.

 

Only in male rats evidence of thyroid effects was found. T3 and TBC were increased in the high dose group as well as thyroid weights. The thyroxine-binding capacities (TBC) were increased in males of all dose levels in Week 12, and in the high dose in Week 3. Even though the increases were statistically significant, they were not considered toxicologically relevant, since they were small and the individual animal data lay mostly within the range of reference values for animals of that age. The only observation that was outside the range of reference values was the increase of TBC in individual animals of the 100 ppm treatment group (males), but since this was an isolated finding, it was not considered of biological relevance. Additionally, T3 increased in males in Week 3 already at 25 ppm showing statistical significance. In conjunction with the short-term studies described above, this observation could be an early sign of thyroid alteration as seen in this study (described below and in short term studies). However, the increase was small and not seen in Week 12, where only 1600 ppm led to a statistically significant increase. Therefore, this finding is considered ambiguous. The change of thyroidal parameters is regarded to be a secondary effect to hepatic enzyme induction, especially UDP-GT induction, and higher T4 turnover. All findings on thyroid parameters were fully reversible within the recovery period.

 

The higher protein (males from 400 ppm, females from 100 ppm) and cholesterol blood concentrations (males and females at 400 ppm and higher doses) in Week 3 could be considered a sign of transient effects on fat and protein metabolism. However, this was not confirmed by any other finding.

Regarding the immune system, slightly increased macrophage activation, and the increase of mitogen stimulation (increase of the LPS stimulated cells) in the spleen at 1600 ppm was considered test-substance related but a secondary effect due to other organ-influencing effects of the test substance.

 

With regard to hematological parameters, mean clotting time was decreased in females of the high dose group in Week 13 and 12. The remaining hematological parameters did not show alterations considered to be biologically relevant. The only observation that can be seen as ambiguous is the reduced monocyte count. In females, the monocyte count was reduced in Week 12 from 100 ppm onward. The data for 100, 400 and 1600 ppm show statistical significance but are not dose-dependent, since the 1600 ppm result is higher than the 400 ppm results. Therefore, it can be concluded that the hematological examinations did not give evidence of adverse effects on red and white blood cells.

 

Taken together, subchronic administration of the test substance via the diet resulted in toxicologically relevant effects in rats at 400 ppm and higher doses. Hepatic enzyme induction was seen as the primary effect resulting in macroscopic alterations, histopathological changes in the liver. The effects observed on the thyroid can be considered as secondary toxicological effects. Therefore, the NOAEL was set at 100 ppm (corresponding to 7.3 and 7.6 mg/kg bw/day for males and females, respectively). In the rat, the liver was identified as the main target organ after subchronic dietary administration. But although there were changes in some clinical chemistry parameters (liver enzymes and thyroid hormones) at dose levels potentially relevant for classification according to CLP Regulation, these changes are considered indicative of increased liver and thyroid activity as the result of adaptive changes.

 

Combined 2-year chronic and carcinogenicity study in rats

In a two year study with Wistar rats, chronic and carcinogenic effects of the test substance were assessed (M-003817-02-1). The rats received the test substance in the diet. The dose levels of 25, 50, 500 and 1000 ppm (corresponding to 1.2, 2.5, 25.2 and 51.7 mg/kg bw/day in males and 1.6, 3.3, 33.5 and 69.1 mg/kg bw/day in females) were based on the subchronic toxicity study described above. Groups of 50 female and 50 male rats were investigated over two years, while additional groups of 10 male and 10 female rats were sacrificed after one year (interim sacrifice).

 

The study was conducted according to OECD guideline 453 with deviations and under GLP conditions. The deviations were missing examinations, namely blood and urine were not withdrawn after 3 months and animals were not fasted prior to blood sampling, thyroid and uterus weight were not assessed, no skin histopathology was performed  and the feed was not analyzed for phytoestrogens. However, meets the criteria of a key study and is valid.

 

During the course of the study, no evidence of a test substance-related increase in mortality was found. The incidence of death in control females was rather high. Overall mortalities were 11-12-14-10-9  and 28-22-14-19-17 for males and females, respectively (0-25-50-500-1000 ppm). The low survival rate of control females led to several uncommonly low findings in the observations described below. Also, no clinical signs were recorded in animals treated with the test substance. Body weights were significantly reduced in males and females from 500 ppm onward. This finding was more pronounced in females than in males, with reduced body weight of up to -21%. Feed consumption was only slightly (but statistically significantly) reduced at several time intervals in males and females.

 

Regarding non-neoplastic changes in the eyes, the only finding of the ophthalmological examination at terminal sacrifice was a higher incidence of lens alterations in the cortex/nucleus of females of the 500 and 1000 ppm treatment groups. Histopathological findings in these groups were retinal atrophy and lens degeneration. However, the incidences in controls were rather low and a dose-dependency is not suggested up to 500 ppm. In the 1000 ppm group, sciatic nerve degeneration was found more frequently in female rats than in the corresponding control group. Comparison with historical control data (M-282359-01-1) revealed that sciatic nerve degeneration is a common finding in ageing rats and the frequency observed in females at 1000 ppm, was within the historical control range. It should be noted that there were no effects observed in the eye examination at interim sacrifice (1 year), so that toxicity to the eye occurred at high doses and after long-time exposure only. No oculotoxic effect of the test compound was found in males.

The hematological and histopathological investigations did not provide evidence of a test substance-related injury to the blood, the coagulation, and the blood forming tissues.

 

Liver enzyme induction was seen from 50 ppm in males and 500 ppm in females onward. Several Phase I and II liver enzymes showed enhanced activity compared to control, i.a. UDP- GLU-T. At interim and at final sacrifice, hepatocellular hypertrophy was observed (from 500 ppm onward, at final sacrifice of males also in the 50 ppm treatment group). Histopathology of animals killed after two years of treatment also revealed cytoplasmic change in the hepatocytes (eosinophilic cytoplasm with basophilic strands) in both sexes. In males of the high dose group, increased incidences of hepatocellular vacuolation was observed and in females necrosis and biliary cysts were slightly elevated at 500 ppm and above. Moreover, males treated with 50 ppm or higher doses and females treated with 1000 ppm of the test substance showed increased incidences of mixed eosinophilic-clear cell foci while basophilic foci were reduced in both sexes and clear cell foci in the liver were reduced in females of the high dose group. The histopathological findings are likely linked to induced hepatic enzyme activity of phase I and phase II enzymes at doses of >= 50 ppm in males and of >= 100 ppm in females. Only a few changes of other clinical chemical parameters were observed. There was a tendency of lower activities, especially of ASAT in males and females and APh in males essentially at the highest dose that can be regarded as a possible secondary consequence of the enzyme induction. Cholesterol was increased in both sexes in Week 26 while triglycerides and bilirubin were decreased and protein concentrations were increased. All these findings occurred in males and females at 500 and/or 1000 ppm, with the exception of bilirubin alteration, which was found in male animals only. Macropathological observations support this assessment as liver weights were increased in both sexes (relative weights up to 31% in males at terminal sacrifice, and up to ca. 16% in females at interim and terminal sacrifice).

 

As reported in the short term studies, the thyroid was also affected by treatment with the test susbtance in this study. At interim sacrifice, one male of the high dose group had a follicular cell adenoma. This finding would not be of concern if isolated, however, at final necropsy, there was a significant and dose-dependent increase in follicular cell adenomas of the thyroids in 500 and 1000 ppm males and a very slight increase in 1000 ppm females. Histopathological investigations revealed hypertrophy of thyroid epithelium associated with an increase of colloid clumping in males of the 500 and 1000 ppm treatment groups at interim sacrifice and at 50 ppm and higher doses at terminal sacrifice. In females, this change was found at terminal sacrifice only and at 500 ppm and above. However, these alterations were only minimal to slight. Colloid alteration and pigment in the follicular epithelium were significantly increased in both sexes from 500 ppm on (final sacrifice). Deviating from the results of earlier short-term studies, no alterations in T3 and T4 levels were observed in peripheral blood but TSH was increased in males (500 and 1000 ppm) and females (1000 ppm), which may be an indication that the higher metabolic degradation of thyroid hormones may have been compensated by an increase in thyroid activity. At termination, enzyme levels in liver tissue were not investigated, but liver histopathology confirmed that thyroid findings were only seen at dose levels with liver enzyme induction (M-496853-01-1). Such activation may lead to the adenomas described and the increased incidence of thyroid adenomas may be a secondary effect of hepatic enzyme induction and not caused by a primary tumorigenic effect of the test item.

 

Further indications of secondary effects of the hormone imbalance caused by liver enzyme induction after dietary administration of the test compound, are effects on the female reproductive system. The incidences of ovarian cysts and uterine adenocarcinomas were increased from 500 ppm onward. The incidence of lacteal cysts in the mammary glands was reduced at a dose of 1000 ppm. Moreover, the additional evaluation of the uterus after 1 year of treatment (interim sacrifice) showed that glandular hyperplasia of the uterine mucosa was found more frequently in treated females (500 and 1000 ppm). However, it is not clear whether this finding is related to treatment or to other findings in the uterus like endometritis or stromal polyps which are frequently found in females of this strain and age. Therefore, this finding needs to be assessed as ambiguous.

 

Damage of the nervous system and skeletal muscles at 500 ppm and above in both sexes was observed, although less pronounced in males. In females, increased incidence of cholesterol clefts in the nerve roots of the spinal cord (1000 ppm) was reported as well as radiculoneuropathy (500 and 1000 ppm), skeletal muscle atrophy (500 and 1000 ppm) and degeneration as well as mononuclear infiltrates in the sciatic nerve (1000 ppm). In both sexes, degeneration of the sciatic nerve was observed (500 and 1000 ppm). However, sciatic nerve degeneration is a common finding in aging rats and muscle degeneration appears also spontaneously in untreated rats of high age. Moreover, none of the observations was found at interim necropsy and other studies using even higher doses could not reproduce neurotoxicty (please refer to the respective IUCLID section). Therefore, a primary neurotoxic effect is considered unlikely and the finding may be due to the general metabolic and hormonal imbalance caused by the substance via hepatic enzyme induction, the observed marked morphological changes of the liver and body weight reduction. The observed effects were therefore considered a high-dose phenomenon.

 

Further effects observed were higher incidences of cholesterol clefts between the anterior pituitary and the pars intermedia in males dosed with 500 and 1000 ppm and significantly increased incidences of sinus histiocytosis in the mesenteric lymph node of females at 1000 ppm.

 

Taken together the most sensitive parameter in this study was hepatic enzyme induction, especially of UDP-GT, in males from 50 ppm and in females from 500 ppm onward. Further phase I and II enzymes were also induced as well as the hepatic markers ASAT and Aph in males. Macroscopically and histopathologically, the liver was altered, emphasizing the hepatic effect of the test substance. Secondary effects were thyroidal alterations, both non- neoplastic and neoplastic. Tumors were also found in the uterus along with histopathological and macropathological changes. These findings may be due to hormonal imbalance as a consequence of the described hepatic enzyme induction. Changes in liver enzymes as well as liver pathology in general were more pronounced in males, as were thyroidal tumors. In females the test item induced more pronounced effects on the nervous system and skeletal muscles. However, as discussed above, these were only relevant at the highest dose and can be seen as a high-dose phenomenon. Oculotoxic effect effects were observed in females at the highest dose levels and after life-long treatment only. Even though the test substance may not directly induce cancer, formation of cancer was observed at high dietary doses of the test substance (500 ppm and 1000 ppm). The NOAEL for general toxicity for males was set at 25 ppm and for females at 50 ppm (corresponding to doses of 1.2 mg/kg bw/day and 3.3 mg/kg bw/day, respectively).

 

Conclusion on repeated dose studies in the rat

In all studies, it was shown that treatment with the test substance led to increased hepatic enzyme induction. This in turn led to various secondary effects with alterations in the thyroid being the most prominent one in all studies performed. Due to higher T4 turnover, TSH release was increased, leading to a hyperactivation of the thyroid with macro- and histopathological alterations. This is supported by the fact that alteration in thyroid parameters only occurred at doses where hepatic alterations also were observable.

 

A further secondary target organ was the uterus, where histopathological changes as well as increased incidences of adenomas were found. Apart from that, all alterations were minimal to slight, within the range of historical control data or occurred at high doses (above 50 mg/kg bw/day) after life long treatment.

An additional expert statement (M-496853-01-1) investigated the mode of action of the development of thyroid tumors in male rats and the human relevance both with regard to tumor development and to the hepatic enzyme induction. The mode of action for thyroid tumors will be discussed in the Carcinogenicity part of this dossier. However, the author concluded that all findings in the thyroid and on thyroid parameters were at doses associated with induced hepatic enzyme induction. At these doses, hepatocellular hypertrophy was also observed, always more pronounced in males than in females. From the different studies conducted it is evident that the induction of hepatic enzymes is crucial for the secondary effects observed in rats. It was found by different studies, that human P450 induction is lower than for rats (Silkworth et al., 2005; Waxman, 1999; Xu et al., 2000). Moreover, in rats, the half life of T4 is shorter due to the lack of high affinity thyroxine-binding globulin which is available in humans but missing in rats, leading to facilitated metabolism and excretion of T4 (U.S. EPA, 1998, Hill et al., 1998; McClain, 1989). In addition, T3 has a shorter half-life in rats, which contributes together with the higher turnover of T4 to increased TSH production and release in rats compared to humans. Therefore, the effects seen in rodents are likely not relevant for humans, a classification for specific target organ toxicity - repeated exposure (STOT-RE) according to the CLP regulation is therefore not required.

The mode of action observed and analyzed for the long-term study with the test substance in Wistar rats has been addressed in the Guidance on the Application of the CLP Criteria (version 5.0, 2017) by ECHA. In chapter 3.9.2.5.3. on mechanisms not relevant to humans, it was stated: "Humans, unlike rodents, possess a T4 binding protein that greatly reduces susceptibility to plasma T4 depletion and thyroid stimulation. Thus, such e mechanism/effect cannot be directly extrapolated to humans, i.e. these thyroid effects observed in rodents caused by an increase in hepatic UDPG-transferase are therefore considered of insufficient concern for classification.”

 

Repeated dose toxicity in mice - oral

In mice, two subacute and one subchronic study were performed. The two subacute studies were assessed in a weight-of-evidence approach, while the subchronic study is regarded as a key study.

 

Subacute oral toxicity studies in mice

To assess the subacute effects of the test substance in mice after repeated oral administration, 2 studies are available that can be used in a weight-of-evidence approach.

In the first subacute study, B6C3F1 mice were fed with the substance via the diet at nominal doses of 50, 200, 2000 and 10000 ppm for 14 days (M-000821-01-1). Analysis of the feed and the feed intake determined the actually ingested dose as 21.6, 84.3, 765.1 and 4143.2 mg/kg bw/day for males and 29.8, 113.2, 1201.2 and 5449.8 mg/kg bw/day for females. Groups consisted of 5 animals per sex and dose. The study was conducted to assess effects in the liver, so even though it was performed according to OECD guideline 407, several deviations were noted: the study duration was shorter than recommended, no hematological analysis was performed and clinical chemistry was limited to liver specific markers. Organ weights were only determined for the liver and histopathology was performed for the liver, only.

 

No animals died prematurely due to test substance intake. Body weight and feed consumption were unaffected by treatment but water uptake decreased in males at 2000 ppm and above compared to controls. Liver enzyme induction of phase I and phase II enzymes occurred at 200 ppm in males and 2000 ppm in females. However, in males, the induction was very slight. Even though no gross pathological findings were observed, the liver weights were increased at 2000 ppm and above in both sexes (up to +15.9% absolute and +24.9% relative liver weight in males and +32.6% absolute and +38.7% relative liver weight in females). Histopathological examinations revealed hypertrophy of centrolobular hepatocytes with more amorphous cytoplasm of the affected liver cells in males at 200 ppm (slightly) and above and in females at 2000 ppm and above. At 2000 and 10000 ppm, slightly increased minute vacuolar fat storage was also observed in males and females.

 

All other findings were not treatment-related.

 

Taken together, the test substance induced hepatic effects starting at doses of 2000 ppm in both sexes. These effects were evident as a dose-dependent increase of phase I and phase II enzymes. Related to this induction were increased liver weights in males and females at 2000 ppm and above and histopathological alteration of hepatocytes (starting at 2000 ppm in males and females). The NOAEL for liver enzyme induction was set at 200 ppm (corresponding to 84.3 mg/kg bw/day for males and 113.1 mg/kg bw/days for females).

 

In the second study used in the weight-of-evidence approach, groups of 3 males and female B6C3F1 mice were fed with the test substance at dose levels of 100, 1000 and 10000 ppm for 21 days (M-000688-01-1, 1994). The actual doses ingested were 30.1, 367.8 and 4141.0 mg/kg bw/day for males and 63.9, 559.3 and 5785.1 mg/kg bw/day for females. The study was conducted according to OECD guideline 407 and served as a dose range finding experiment for the subchronic toxicity study. Therefore, the study procedure deviated from the guideline in several aspects: The treatment duration was too short, no hematology or clinical chemistry analysis were performed, histopathology was not assessed, organ weights were only determined for liver and kidney and the eyes were not examined.

 

No mortalities occurred during the testing period and clinical signs of control and treatment animals were comparable. At 10000 ppm, mean body weight was reduced in males up to -8.3% compared to controls. Feed consumption was reduced in females at 1000 ppm and higher doses and increased in males at 10000 ppm compared to control animals, while water consumption was decreased in females of the high dose group only. The liver was enlarged in 2/3 males in the high dose group and absolute and relative liver weights were increased in both sexes at 1000 ppm and above. The slight decrease in relative kidney weights in females of the high dose group compared to controls was considered incidental as it was a small change only (5%) and the number of test animals was not sufficient to determine this as a treatment-related effect.

 

In summary, in this study the test material exhibited effects at 1000 ppm and higher doses in male and female mice, as seen by altered body weight, feed and water consumption as well as altered liver weight and macropathological appearance. The kidney was not affected by treatment. Therefore, the NOAEL was set at 100 ppm (corresponding to 30.1 mg/kg bw/day for males and 63.9 mg/kg bw/day for females).

 

 

Subchronic oral toxicity study in mice

For the assessment of subchronic toxicity in mice, one key study is available (M-000697-02-1, 1995). This study also served as a range finding study for the chronic carcinogenicity study described in the respective section of the dossier.

 

Groups of 10 male and 10 female B6C3F1 mice received the test substance at dose levels of 50, 250, 1250 and 6250 ppm in the diet for 14 weeks. The actually ingested doses were 19.9, 102.6, 542.4 and 2819.9 mg/kg bw/day for males and 27.2, 139.1, 704.3 and 3351.0 mg/kg bw/day for females. The study was conducted under GLP conditions and according to OECD guideline 408 with some deviations from the current guideline. Especially the thyroid parameters were not assessed (thyroid weight, TSH, T3 and T4 levels). Moreover, the estrus cycle of female mice was not investigated, prostate, uterus, thymus, spleen and pituitary weight were not determined. These deviations were considered acceptable and the study was considered valid and reliable.

 

Daily inspection of the animals gave no indication of treatment induced effects on health, altered behavior or mortality in the whole dose range tested. Body weight was reduced in males of the high dose group (up to -14%) compared to controls. Feed consumption was slightly increased in males at 1250 and 6250 ppm, suggesting decreased feed efficiency. In males and females of the high dose group, water intake was decreased compared to controls. However, for females this was not considered relevant since no other toxicological effects occurred.

 

Analysis of blood and liver tissue samples showed hepatic effects in males at 250 ppm and above and in females from 1250 ppm onward. N-demethylase activity was increased in male and female mice compared to controls. In both sexes, bilirubin concentration was decreased and cytochrome P450 content increased at 1250 ppm and above. In the high dose group, decreased cholesterol (males, females), increased triglycerides (males) and slightly reduced protein and albumin concentrations (females) occurred. Associated with these findings were increased absolute and relative liver weights in both sexes from 1250 ppm onward and hepatocellular hypertrophy in males at 250 ppm and above and in females at 1250 ppm and higher doses. The marginally, but statistically significantly decreased hematocrit values compared to controls at 1250 (males) and 6250 ppm (males, females) were seen as a secondary effect to hepatic enzyme induction.

 

No toxicologically relevant effect was seen in the kidneys. In the histopathological assessment, a reduction/loss of sex specific vacuoles in epithelial cells of the proximal tubule in males of the 1250 and 6250 ppm groups as well as slightly lower kidney weights are regarded most probably as an unspecific response to physiological stress. In females, no sign of kidney effects was observed. The adrenal weights of females were slightly (but statistically not significantly) increased at ≥ 1250 ppm. This correlated with an enlargement of the adrenal X zone which was microscopically already visible at lower doses (≥ 50 ppm). The ovaries appeared to have lower numbers of old corpora lutea at 1250 and 6250 ppm.

 

All other findings occurred sporadically, were not dose-dependent and/or within the range of historical control data.

 

Following dietary administration of the test substance over 14 weeks, male and female mice exhibited test substance-related effects mainly on the liver, as seen as increased liver weights, hepatocellular hypertrophy and liver enzyme induction. These effects occurred at 250 ppm and higher doses in male mice, and at 1250 ppm and higher doses in female mice. The NOAEL was set at 50 ppm for males (corresponding to 19.9 mg/kg bw/day), no NOAEL for females could be established as effects were already present at the lowest dose level. For female mice, the LOAEL was set at 50 ppm (corresponding to 27.2 mg/kg bw/day) based on vacuolisation, hypertrophy and enlargement of the adrenal X-zone. However, since the adrenal X-zone is not present in humans, this finding is not considered relevant for human toxicological assessment.

 

 

Conclusion on repeated dose studies in mice

From the two subacute and one subcronic study in mice it can be concluded that hepatic enzyme induction also occurs in this species, as already seen for rats. Related to this are macro- and micropathological alterations of the liver as increased liver weight and hepatocellular hypertrophy. The thyroid and thyroid parameters were not assessed in mice. However, as stated above, a relevance for humans is not assumed and based on the available data, no classification for specific organ target toxicity - repeated exposure (STOT-RE) according to CLP regulation is required.

 

 

Repeated dose toxicity in dogs - oral

For assessment of repeated-dose toxicity of the test material to dogs, two key studies are available (one subchronic, one chronic) that were conducted according to OECD guideline 409 and 452, respectively, and under GLP conditions.

 

Subchronic oral toxicity study in dogs

 

In the first study, groups of 4 male and 4 female Beagle dogs were administered the test substance in the diet for 13 weeks (M-003814-01-1, 1988). Initially, the doses were set at 250, 1000 and 4000 ppm based on a previously conducted subacute oral toxicity study in dogs in which the test substance was administered via the feed at dose levels of 100, 300 and 1000 ppm to male and female Beagle dogs over 4 weeks (M-003816-02-1). In this study, the 1000 ppm dose was increased to 2500 ppm during the study and an additional group received 2500 ppm in their feed throughout 4 weeks. No relevant toxicological findings were observed except for a slight decrease in feed intake. In the present study, the highest dose had to be reduced due to severe clinical signs including vomitus, slight tremor, reduced or no food intake, and body weight decrease within the first days of treatment. Therefore, the high dose animals were fed with the plain diet from Day 5 to 15 allowing the animals to recover. Treatment was afterwards continued with 2000 ppm of the test substance in the diet. The duration of the study was prolonged for two weeks so that the high dose animals received 2000 ppm of the test substance for 13 weeks.

 

The feed was analyzed for the concentration of the test substance in the diet and the actual doses received over the course of the study were 8.5, 34.9 and 68.0 mg/kg bw/day for males, and 8.9, 34.7 and 65.3 mg/kg bw/day for females. All animals were checked daily for signs of ill health, body weight and feed consumption were assessed weekly as well as water consumption. Before beginning of treatment and in weeks 7 and 15, ophthalmoscopic examinations were performed. Blood was withdrawn before the study and in weeks 2, 7 and 15 to determine hematology and clinical chemistry. Electrocardiograms (ECG) were performed two weeks before start of treatment and in weeks 7 and 15 (before and 2h after administration), At sacrifice, animals were subjected to a gross necropsy, a liver sample was taken for investigation of hepatic enzyme induction, several organs were weighed and histopathology was performed.

No animals died during the course of the study. As mentioned above, animals that were fed with 4000 ppm of the test substance started vomiting after the first administration, and reduced feed consumption or refused feed intake were observed. After recovery, 2000 ppm of the test substance was accepted, but feed intake was slightly reduced in males and females compared to controls during the first half of the study. In the mid dose groups, reduced feed intake was also observed in two females at several intervals. Body weight was reduced in males of the high dose group only and body weight gain was slightly reduced (-12.8%). Treatment with the test substance did not have an influence on water consumption, ophthalmological, hematological or urinalysis findings.

 

Regarding clinical chemistry, several enzymes in plasma and liver samples were altered. Increased glutamate dehydrogenase (GLDH) levels were found in one female in Week 2 (250 ppm), however no dose-relationship was observed and this was seen as an adaptive response. At 1000 ppm, ALAt activity was increased in one male in Week 2 and at 2000 ppm ALAT activity was increased in two males and one female. In addition, at 2000 ppm, GLDH activity was increased in one male and one female. N-demethylase activity was also increased slightly at this dose level. All these changes were reversible during the study period and are seen as a sign of metabolic induction. At 2000 ppm, slightly but statistically significantly increased epoxide hydrolase EH (males), marginal but not significant induction of Glutathione-S-transferase (GS-T) and UDP-GT (females) and reduced 7-Ethoxyresorufin deethylase (EROD) activity (males, females) were found. All these findings suggest metabolic induction as do the slightly increased liver weights. The slight increase of UDP-GT might also explain decreased T4 and increased TSH levels (as seen to a more pronounced extend in rat studies). However, none of these changes led to an adverse effect and they were considered rather to be a result of increased metabolic activity caused by the test substance (as is common for many xenobiotics).

 

The prostate was altered both in terms of organ weight and histopathologically in the high and mid dose groups. In all male animals of both groups, slight to moderate hypertrophy of the prostate occurred and the mean absolute and relative prostate weights were increased. This finding is considered ambiguous since such changes are known to show a wide biological variation with respect to severity and incidence in young mature dogs.

 

Under the conditions of the study, the test substance induced altered feed consumption and body weight in treated animals compared to controls at 1000 ppm and higher doses. Altered liver enzyme activity was observed indicating increased liver metabolization. This is also seen by slightly increased liver weights at necropsy. Since this can be seen as an adaptive response and no other adverse effects were observed, it is not considered of toxicological concern. The prostate was altered both in terms of organ weight and histopathologically in the high and mid dose group of males. Based on these results, the NOAEL was set at 250 ppm (corresponding to 8.5 mg/kg bw/day) for male dogs and 2000 ppm (65.3 mg/kg bw/day) for female dogs.

 

Chronic oral toxicity study in dogs

The second study in dogs was performed under GLP conditions according to OECD guideline 452 (M-003818-01-1, 1998). Groups of 4 male and 4 female Beagle dogs received the test substance via the diet for 52 weeks at doses of 40, 100, 250 and 1000 ppm (corresponding to 1.42, 3.60, 8.88 and 34.42 mg/kg bw/day for males and 1.39, 3.44, 8.30 and 33.80 mg/kg bw/day for females). Additional groups of 3 male dogs received the test substance via the feed for only 26 weeks, the doses were 100 and 1000 ppm (corresponding to 3.23 and 32.21 mg/kg bw/day). For both treatment durations, animals of the control group received the plain diet. The dose selection was based on the subchronic feeding study (M-003814-01-1) in Beagle dogs over 13 weeks described above as well as an additional subacute study addressing palatability. In the latter study, dogs were administered the test substance by capsules at doses of 95 and 152 mg/kg bw/day. Severe signs of toxicity, like vomitus, reduced feed intake, increased salivation, tremor and ptosis occurred at both doses in a dose-dependent manner. 1 animal in the high dose group died and plasma concentrations on Day 1 and Day 12 showed that concentrations of the test substance on Day 12 were higher than on Day 1, indicating a building-up of the test substance. Based on these two studies, the concentrations in feed in the present study were set as follows: 40, 100, 250 and 1000 ppm for 52 weeks. Additionally, groups of three male dogs received 100 and 1000 ppm for 26 weeks to analyze changes in the prostate over a prolonged period of time.

All animals were checked daily for signs of ill health, body weight and feed consumption were assessed weekly as well as water consumption. Before beginning of treatment and in weeks 6, 14, 26, 39 and 52, ophthalmoscopic examinations were performed. Blood was withdrawn before the study and in weeks 6, 13, 26, 39 and 52 to determine hematology and clinical chemistry. Electrocardiograms (ECG) were performed as well as analysis of pulse rates, heart rates and reflexes several times during the study. At sacrifice, animals were submitted to gross necropsy, a liver sample was taken for investigation of hepatic enzyme induction, several organs were weighed and histopathology was performed. The prostate of animals killed at interim and terminal sacrifice was assessed thoroughly since signs of prostate alterations has been found in the subchronic study described above.

 

No mortality occurred during the study. In addition, no differences in heart and pulse rates, ECG measurements, nutritional state and clinical signs were observed between treatment and control groups. Body weight and body weight gain were comparable across all groups. In females, feed intake was slightly reduced in the 1000 ppm group. Ophthalmoscopic and hematological investigations did not show any differences between control and treatment groups and no changes were seen in the urinalysis.

 

In both satellite groups (100 and 1000 ppm, males), T4 levels were slightly reduced, while TBC and T3 levels remained unaltered. T4 levels were still within the historical 2s-range, and this change was not found in the corresponding main groups after 52 weeks and no microsomal liver enzyme induction was seen after 52 weeks of treatment. Therefore, the finding was considered incidental. At interim sacrifice, liver weight was increased slightly and a hepatocellular cytoplasmic change was seen in males of the high dose group (1000 ppm). Since this was not observed after one year of treatment, the finding is considered a result of temporary adaptive response as known for other xenobiotics.

The prostate was unaltered after 26 weeks of treatment but prostate weight and size were found to be slightly altered after 52 weeks. Morphological investigation including sonography of the dog prostates did not reveal a treatment-related effect. Additionally, histopathological examination of the prostate of dogs treated for 26 and 52 weeks did not reveal any alterations, as compared to the control animals. Therefore, adverse effects on the prostate by the test substance are unlikely and the changes in size and weight observed are considered a consequence of high spontaneous variation.

 

Taken together, the test item caused a slight change of feed intake and liver weight in the highest dose groups after 52 weeks of administration. Adverse effects on the liver and prostate observed in the subchronic study in dogs were not. Also clinicochemical investigations did not show effects on hepatic enzyme induction or thyroid impairments as seen in rodent studies. Therefore, a dose of 250 ppm (corresponding to a dose of 8.88 mg/kg bw/day) is regarded as the NOAEL for dietary administration of the test substance to the male Beagle dog over 52 weeks. No adverse effects were observed up to the highest dose in female dogs (NOAEL >= 1000 ppm corresponding to 33.8 mg/kg bw/day). The study was conducted under GLP conditions according to OECD guideline 452 and is considered valid.

 

Conclusion on repeated dose studies in dogs

In both the subchronic and chronic study, no effect on the thyroid could be detected. Altered T4 blood concentrations were found sporadically but remained within the historical control range as shown in a re-evaluation of the existing thyroid data (M-496983-01-1). They were therefore not considered biologically relevant. Moreover, no alterations of thyroid morphology or histopathology were found. The prostate was slightly altered in size and weight without sonographical or histopathological correlates indicative of an adverse effect. The main effects observed in Beagle dogs were reduced feed consumption, reduced body weights, increased liver and prostate weights and hepatocellular cytoplasmatic change.

 

Inhalation repeated dose toxicity

Subacute inhalation toxicity study in rats

In the 28 day repeated-dose inhalation study (M-241815-01-1, 1998), Wistar rats were exposed to the test substance at concentrations of 2, 20 and 200 mg/m³ (nominal concentration, dust), under dynamic directed-flow nose-only exposure conditions. However, due to severe clinical signs in the high dose group, the highest concentration had to be reduced to 100 mg/m³ air. Analytical analysis of the test atmosphere gave concentrations of 2.0, 18.2 and 142.2 mg/m³ (mean over entire testing period). Groups consisted of 10 animals per sex and dose and exposure lasted 6 h/day for 5 days per week. The doses were based on an acute inhalation study and a 1-week subacute pilot study in rats. In the acute inhalation study, rats were exposed to the test substance at concentrations of 80, 481, 1523, and 2535 mg/m³ air. The NOAEC was found to be 80 mg/m³ air for males and females and the LC50 > 2535 mg/m³ for males and approximately 1223 mg/m³ in females. In the subacute pilot study (M-000725-02-1), rats were exposed to the test substance at 1.97, 19 or 205 mg/m³ air. Only minor influences on the respiratory tract were found up to the highest concentration tested. At 205 mg/m³ air, liver weight was increased transiently and hepatic enzyme induction was found. At 19 mg/m³, grip strength was increased transiently and an increased incidence of darker spleens in the female rats of this group was found. All findings were reversible within 2 weeks. 19 mg/m³ was therefore seen as the lowest effect level. Based on these results, the concentrations for the main subacute study were chosen to be ca. 2, 20 and 200 mg/m³ air. 2 mg/m³ was expected to be non-toxic while 20 and 200 mg/m³ air should induce toxic effects. During the course of the study (in week 2), 200 mg/m³ air had to be reduced to 100 mg/m³ air to prevent undue distress of animals or even mortality.

 

The test atmosphere was generated using a Wright Dust Feeder. The aerosol was highly respirable to rats, with an average Mass Median Aerodynamic Diameter (MMAD) was ca. 2.9 µm. The percentage of particles below 3 µm was 54.5 (2 mg/m³), 51.6 (20 mg/m³) and 53.1% (200 mg/m³). The Geometric Standard Deviation (GSD) was ca. 1.8. No mortality was observed in the study but as already described, the highest concentration had to be lowered to 100 mg/m³ in the second week due to severe clinical signs. These were bradypnea, labored breathing pattern, reduced motility, piloerection, ungroomed hair-coat, atony, rales, salivation, mydriasis, miosis, tremor and vocalization. Moreover, body weight and rectal temperature were altered when rats were treated with 200 mg/m³ air. It was concluded that most of the signs predominantly related to respiratory distress rather than specific central nervous effects (mydriasis, miosis, tremor, body weight, decreased rectal temperature) which subsided completely after reduction of the test concentration. The assessment of reflexes did not reveal any differences between the groups. The only sign observed was that on Day 2, some rats of the high level exposure group experienced a decreased tonus and light reflex (reversible).

 

Hepatotoxic effects occurred in both sexes, but more pronounced in females. These manifested in increased hepatic enzyme activity in males at 20 (N-demethylase activity increased, although only slightly) and in both sexes at 200 mg/m³ (N- and O-demethylase activity increased, higher levels of cytochrome P450, increased APh in females). Other clinical chemistry parameters were also altered in the high dose group, namely increased phosphate in males, increased glucose in males and females, increased cholesterol, bile acids and calcium in females. These findings suggest an early onset of hepatic toxicity. This is also reflected in gross pathological and histopathological findings. The absolute and relative (to body) liver weights of males and females in the high dose group were increased by 12.2 to 16.8%. In the mid (males) and high (males, females) dose groups, slight hepatocellular hypertrophy was observed. Males of the high dose group also showed hypertrophy of the thyroidal follicular epithelium.

 

No effects were seen in ophthalmological and urine investigations. All other findings were considered incidental and not treatment related.

 

In summary, the data generated in this study reflects the findings of other repeated dose investigations with the test material. The test substance had an influence on liver enzyme induction, leading to macroscopical and histopathological changes of the liver. Signs of hepatotoxicity comprised liver enzyme induction, minimal to slight hepatocellular hypertrophy and increased liver weights observed at the highest tested concentration. Furthermore, slight thyroid follicular cell hypertrophy also was noticed. No adverse effects on the lungs and extrapulmonary organs were found. The NOAEC for the test substance after repeated inhalation exposure for 28 days is 18.2 mg/m³ for males and females.

The study was conducted according to GLP conditions and according to OECD Guideline 412. Since the study was conducted in 1998, there are deviations to the current guideline: Blood was taken from non-fasted animals for glucose determination, no BALF analysis or determination of lung burden was conducted, and humidity in the exposure chamber was lower than recommended. These deviations are considered minor and the study is valid and a key study for the assessment of repeated-dose toxicity.

 

 

Dermal repeated dose toxicity

Subacute dermal toxicity study in rats

 A key study assessing the potential of the test substance to cause toxicity following repeated dermal application is available (M-000824-01-1, 1997). 5 rats/sex/dose received 100, 300 or 1000 mg/kg bw test substance/day for 28 days. Two additional groups (control and high dose) with 5 rats/sex/dose were included as recovery groups and observed for 2 weeks after the end of the treatment. The test substance was moistened with water and applied to over 10% of the rat’s skin for 6h/day. During this time, animals wore a jacket to prevent them from ingesting the test item. The treatment was repeated 5 times/week in the first 3 weeks and daily in the fourth weeks (22 days of treatment). The control group was exposed to the vehicle only.

No mortality occurred during the course of the study and treated animals did not show any treatment-related clinical signs. The skin redness and skin thickness at the application site did not change significantly and was comparable between control and treated animals. The body weight of the treated animals was not significantly affected during the treatment period. A slight body weight loss during the first week (8%) in the high dose females was considered to be due to a reduction in food intake during the same period. The females of the high dose group showed a significant decrease in feed consumption (50%) during the first week of treatment. This could be treatment-related or a sign of stress caused by the treatment rather than the test substance, but as the change was transient it is not considered to be toxicologically relevant. The results of the hematology and clinical chemistry parameters were comparable between control and treatment groups.

The liver was affected by the test substance treatment in males and females. Both the absolute (12.7/13.5%, m/f) and relative (17.2/11.9%, m/f) liver weight was increased in males and females of the high dose group. At histopathology, hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm was observed in males at 300 (3/5 males) and 1000 mg/kg bw/day (5/5 males), and in 3/5 females at 1000 mg/kg bw/day. In the females, the observed hepatic findings were reversible within the 2-week recovery period. In males, the incidences of histopathological changes decreased to 2/5 but did not get back to control levels. The changes are considered toxicologically relevant.

In the thyroid, no gross pathological changes were observed but hypertrophy of the follicular epithelium was observed in both sexes at 1000 mg/kg bw/day (3/5 males, 2/5 females). As in the hepatic observations, the histopathological changes were fully reversible for females and partly reversible for males (1/5 males still had hypertrophy of the follicular epithelium after the recovery period). The changes are considered toxicologically relevant.

 

The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused liver and thyroid changes when applied dermally for four weeks in males and females. The changes in liver and thyroid were fully reversible for females but only partly reversible in males within the two-week recovery period. The observations of this study are in line with the previously reported oral toxicity studies that show an effect of the test substance on hepatic enzyme induction, leading to secondary changes in the thyroid. As no dermal irritation was seen, the NOAEL for local skin effects is 1000 mg/kg bw/day. The NOAEL for systemic effects is 100 mg/kg bw/day for males and 300 mg/kg bw/day for females.

 

Conclusion on repeated dose toxicity

In the repeated dose toxicity studies in rodents the main target organ was the liver. A dose-dependent microsomal liver enzyme induction occurred in rats and mice which was also reflected by histopathological findings. Histopathologically, hepatocellular hypertrophy with a finely structured cytoplasm of liver cells was detected. As a consequence also the liver weights were increased at doses which caused a liver enzyme induction. As a consequence of the liver enzyme induction secondary effects in thyroids were seen in rats as known from many other liver enzyme inducers. Besides changes in T4, T3 and TSH levels also thyroid weights were increased and morphological changes found at respective doses. The toxicological profile was comparable after different routes of administration, i.e. liver enzyme induction with related morphological changes after higher doses and longer treatment were not only seen after oral, but also after inhalative and dermal administration.

In dogs the liver was also the main target, but the effects were weaker than in rodents. Slight morphological effects on the liver and on the prostate were seen after higher doses in the pilot studies and liver effects also at interim necropsy in the chronic toxicity study. Although evidence of an enzyme induction was also observed in dogs, it was less pronounced than in rodents. 

 

In the chronic toxicity and carcinogenicity study in rats at doses of 500 ppm and above growth retardation was observed, especially in females with a more pronounced growth retardation with large body weight differences to controls after 1000 ppm in both sexes. Therefore, this dose clearly exceeded MTD criteria especially in females and caused findings which are related to high-dose phenomena. Main target was the liver with an induction of phase I and phase II enzymes at doses of ≥ 50 ppm in males and of ≥ 100 ppm in females. Also morphological liver changes were observed which were most obviously related to chronic liver enzyme induction, like hepatocellular hypertrophy, cytoplasmic changes in the hepatocytes, increases and decreases of incidences of certain altered hepatocellular foci. The liver weights were increased at respective doses. These changes are considered adaptive, not adverse effects. The primary effect on the liver caused the described secondary effects. As a further consequence of the enzyme induction, the incidence of follicular cell adenomas was increased in 500 and 1000 ppm males and, very slightly, in 1000 ppm females and the incidence of uterine adenocarcinomas at doses of ≥ 500 ppm. These effects are further discussed in the section on carcinogenicity.

 

The mode of action observed and analyzed for the repeated-dose studies with the test substance in rodents has been addressed in the Guidance on the Application of the CLP Criteria (version 5.0, 2017) by ECHA. In chapter 3.9.2.5.3. on mechanisms not relevant to humans, it was stated: "Specific thyroid toxicity via liver enzyme induction:

Certain chemicals cause induction of liver enzymes and are interfering with the regulation of thyroid hormones. An increase in the activity of hepatic UDP-GT results in increased glucuronidation of thyroid hormones and increased excretion. It is known that rodents are highly sensitive to a reduction in thyroid hormone levels (T4), resulting in thyroid toxicity (e.g. hypertrophy, hyperplasia) after repeated stimulation / exposure of this organ. This in turn is related to an increase in the activity of hepatic UDP-GT. Humans, unlike rodents, possess a T4 binding protein that greatly reduces susceptibility to plasma T4 depletion and thyroid stimulation. Thus, such a mechanism/effect cannot be directly extrapolated to humans, i.e. these thyroid effects observed in rodents caused by an increase in hepatic UDP-GT are therefore considered of insufficient concern for classification."

This assessment is supported by the results of the repeated-dose studies conducted in dogs.

Therefore, no classification with regard to specific target organ toxicity - repeated exposure according to CLP regulation is required for the substance.

Justification for classification or non-classification

An extensive data base exists to assess repeated dose toxicity following the oral, inhalation and dermal route. Based on the available data on short- and longterm exposure, the test substance does not meet the classification criteria in regard to STOT-RE for neither route according to the CLP regulation (EC) No. 1272/2008 which is in line with the harmonized classification of the test item according to Annex VI of the CLP Regulation (EC) No. 1272/2008 (Index no. 613-325-00-3).