Reaction mass of bis(oxiran-2-ylmethyl) terephthalate and tris(oxiran-2-ylmethyl) benzene-1,2,4-tricarboxylate

Endpoint summary

Description of key information

Based on the effects observed in a 28-day repeated dose oral toxicity in rats, the no observed adverse effect level (NOAEL) was establised at 75 mg/kg/day in both sexes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-21 to 2014-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: 8 - 9 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 152 - 183g; females: 114 - 144 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1526)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fiber bedding (lot no. 030713).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Route of administration:

oral: gavage

Details on oral exposure:

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Animals showing pathological signs before
the first administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange. Before the first
administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration of Doses
The test item and control formulation were administered at a single dose to the animals by oral gavage at an application volume of 5 mL/kg bw.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulation Analysis
For determination of the concentration of test item in dosing formulations, samples of at least 25 mL were retained from all groups once weekly
during the treatment period and stored between -15 and -35 °C. In total 16 samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period. From the LD and HD group samples of dosing
formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C. In total 4 samples.
In the 1st and 4th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared HD
and LD formulations and stored between -15 and -35 °C. In total 12 samples.
Each sample was retained twice (sample A, sample B, each of at least 25 mL).
At the end of the treatment period all A samples of dosing formulations was analysed at BSL BIOSERVICE Scientific Laboratories GmbH in accordance
with GLP under the reference number 137239.
The B samples were retained at BSL until the analysis had been performed, and were discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item or vehicle on 7 days per week for a period of 14 days.

Frequency of treatment:

The animals were treated once daily.

Remarks:

Doses / Concentrations:
25 mg/kg bw, 75 mg/kg bw and 200 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

40 animals (20 males and 20 females) were included in the study (5 male and 5 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Section schedule rationale : alle male animals together and all female animals together

Observations and examinations performed and frequency:

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the
treatment period. Food consumption was measured weekly during the treatment period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 28 days.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated
effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the
treatment period.

Functional Observations
Once before the first exposure and once in the fourth week of exposure multiple detailed behavioural observations were made outside the home
cage using a functional observational battery of tests. These tests were conducted in all animals.

Haematology
Haematological parameters were examined at the end of the treatment as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

Blood Coagulation
Coagulation parameters were examined at the end of the treatment as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in citrate tubes.

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.

Urinalysis
A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals.

Sacrifice and pathology:

Pathology
One day after the last administration (study day 29) all animals of the treatment period were sacrificed using anesthesia (ketamine, Pharmanovo,
Lot No: 24644, expiry date: 30 June 2015 and xylazin, Serumwerk, Lot No. 00513, expiry date: 31 May 2015) and subjected to a detailed gross
necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and theircontents.

Other examinations:

Organ Weight
The wet weight of the organs of all sacrificed animals was recorded as soon as possible. Paired organs were weighed together.

Histopathology
The organs were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for the animals of the groups 1 and 4 sacrificed at the end of the treatment period.
As requested by the sponsor, additional histology processing was performed of the liver, brain, spinal cord and epididymides of all animals of the LD and MD groups. Histopathology evaluation was performed of all processed organs.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in
the organs of all dose groups. The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory
AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at
the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology).

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. Due to the small size of groups a
statistical evaluation of the results was not performed in this study.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

for details see below

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Observations
Clinical signs like moving the bedding and slight to severe salivation were observed in all dose groups during the second half of the treatment
period and at the medium and high dose levels also during the second treatment week. These signs, observed in close association with oral
application by gavage, are assumed to be related to local effects caused by the bolus administration of the test item formulation and are not
considered to be signs of systemic toxicity. These signs were also observed in control animals on single days and are also partly associated with
the vehicle (DMSO/PEG 400) used in this study.
Diarrhea was noted in 1/10 animals of the HD group, 4/10 animals of the MD group and 3/10 animals of the LD group. It was observed on single
or on 2 consecutive days during mid of treatment. At the end of the treatment period diarrhea was not observed. Although this might be
associated with the test item, it is not assumed to be toxicologically relevant.
On study day 15 respiratory sounds were noted in female animal no. 37 of the HD group. This animal had recovered on the next day. These
respiratory sounds were possibly related to slight lesions caused by the gavaging cannula.
Nasal discharge observed in female LD animal no. 30 on day 15 and in male control animals no. 1 (day 22) and 5 (day 24) are assumed to be
isolated and incidental mild clinical findings.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Body Weight Development
Throughout the treatment period, body weights of control animals were within the normal range of variation for this strain.
In both males and females, the mean body weight increased with the progress of the study in the C, LD, MD and HD groups.
 
Male animals of all dose groups gained between approx. 16 and 30 % less body weight, when compared to controls, independently of dose. The
difference to controls was statistically significant at the LD and HD levels but not in the MD group. It should be noted that a considerably lower
body weight gain was noted at the high dose level especially during the last treatment week (50 % of control value).
At the end of the treatment period, body weights of male animals in LD, MD and HD groups were 10, 8 and 9 % lower, when compared to controls.
However, in LD and MD groups the body weights were already between 4 and 5 % lower on the first day of administration. Thus, the test material is considered to have an attenuating effect on body weight development on male animals only at the high dose level.

Food Consumption
In correlation to the lower body weight of dosed male animals, food consumption was slightly lower in the respective groups, when compared to
controls. In HD group food consumption was considerably lower than in controls during the last treatment week (approx. 40 % of controls).
No effect of the test material on food consumption of female animals was observed.

Haematology and Blood Coagulation
Oral treatment with the test material lead to slightly lower levels of red blood cell parameters analyzed at the end of the treatment period.
Female animals were more prominently affected. Statistically significantly lower levels of red blood cells (RBC), haematocrit (Hct) and haemoglobin
(Hb) were observed at the HD group, when compared to controls (14 %, 16 % and 15 % below controls, respectively). In male animals only a tendency
towards lower values was observed at this dose level (7 %, 6 % and 8 % below controls, respectively), with a statistically significantly lower Hb
concentration, compared to controls. This effect was also observed in female animals at the low dose level. Hct and Hb values were slightly but
statistically significantly lower in this group when compared to controls. The respective values were at the lower border of the historical control
ranges.
Reticulocytes were slightly – but statistically significantly higher in female animals of the HD group when compared to the respective controls.
A slightly but statistically significantly higher rate of monocytes in male animals of the HD group is not assumed to be toxicologically relevant, as the difference in absolute monocyte count was negligible.
Besides, all haematological parameters were within the normal range of variation and no considerable differences were found between control and
dose groups.
The test material had no effect on coagulation parameters determined at the end of the treatment period of this study.
 
Clinical Biochemistry
A high serum level of hepatotoxicity markers alanine aminotransferase (ALT; approx. 7.5 fold of controls) and ASAT (aspartate-aminotransferase,
2.2 fold of controls) was determined in male animals no. 17 of the HD group at the end of the treatment period. This was associated with a high liver
weight and centrilobular hepatocellular vacuolation and inflammatory cells in the liver. Although this was observed in 1/10 animals of the HD group, a relation to the test item cannot be excluded.
At the end of the treatment period serum level of total bilirubin (TBIL) was slightly and statistically significantly higher at the high dose level when
compared to controls (approx. 40-50 % above controls). At the medium dose level female animals were also slightly affected (approx. 36 % higher
than controls). As TBIL serum levels were within the range of historical control data, these statistical significant differences are not assumed to be
biologically relevant.
A slight but statistically significantly higher serum potassium level in male animals of the HD group is not assumed to be toxicologically relevant as
the levels were within the range of historical control data.
Statistically significantly lower ASAT levels in female animals of the LD and MD groups (82 % and 75 % of controls, respectively) are not assumed to be toxicologically relevant, as a slight decrease in this liver parameter is generally not associated with hepatotoxicity. Similarly, statistically significantly
lower creatinine (Crea) level in female animals of the LD and MD groups (78 % and 76 % of controls, respectively) is not assumed to be toxicologically
relevant, as a slight decrease in this kidney parameter is generally not associated with nephrotoxicity. Due to the slight differences in these
parameters, compared to controls, even a relation to the test item is doubtful.
Total protein (TP) was slightly but statistically significantly lower in female animals of the LD group when compared to controls (approx. 91 % of
controls). This is assumed to be an incidental finding. Moreover, as the values were within the range of historical control data, this finding is not
assumed to be of biological relevance.
A slight but statistically significantly lower serum albumin (Alb) level in male animals of the LD group (92 % of controls) is assumed to be incidental
and not biologically relevant, as the values were within the range of historical control data.
Besides, all remaining parameters of clinical biochemistry were within the normal range of variation for this strain and there were no statistically
significant differences between dose and control groups of this study.

Urinalysis
At the end of the treatment period there were no conspicuous findings in urinary parameters of the treated animals. No
considerable differences were found between dose group and control group animals.
 
Pathology
Few specific gross pathological changes were recorded in male and female animals and were not considered to be treatment-related.
Yellow foci on the epididymis were observed in single animals of all groups. This finding is a commonly seen background finding in control animals
and is not assumed to be related to administration of the test material.
Small sized prostate and epididymis and testes were observed in animal no. 20 of the HD group. As this is an isolated finding, it is assumed to be
incidental. This animal also had a dark red pancreas.
Dilated pelvis were observed unilaterally in kidneys of 2/5 male animals of the LD group. As this was also observed in a control animal, it is not
assumed to be toxicologically relevant.
Single findings that are assumed to be incidental were: small-sized urinary bladder and dark red discoloured axillary lymph nodes in 1/5 animals of
the LD group, respectively.
Dark red axillary lymph nodes, large intestines and thymus were observed in female animal no. 33 of the MD group. As these findings were not
observed in other animals, they are not assumed to be related to the test item, but rather appear to be incidental.

Organ Weight
A slightly but statistically significantly increased absolute liver weight (approx. 11 % above controls) was observed in female animals at the high dose
level, when compared to controls. This difference was also statistically significant when related to brain weight (approx. 13 % below controls), however not when related to body weight (approx. 7 % below controls). Although this slight increase in the mean liver weight was only observed in female
animals of the HD group, centrilobular hepatocellular hypertrophy was found histomorphologically in both genders of this dose group. An isolated
high liver weight in animal no. 17 of this group was associated with high levels of serum markers of hepatotoxicity (ALAT, ASAT) and centrilobular
hepatocellular vacuolation and inflammatory cells.
Spleen weight of female animals of the HD group was moderately but statistically significantly higher than in controls (absolute weight by approx.
35 %, relative weight to brain weight by approx. 37 % and relative weight to body weight by approx. 29 %). Spleen weight of male animals, when related to their brain weight, was also dose-dependently slightly higher than in the respective controls (LD group 13 %, MD group 26 % and HD group 32 %
above controls). The differences were statistically significant at the medium and high dose level. Increased spleen weight of female animals is possiblyrelated to a reaction towards changes observed in red blood cell parameters observed in the HD group (see 12.6). Histopathologically, however, no
abnormalities were observed.
In male animals of the HD group combined absolute weight of seminal vesicles, coagulating gland and prostate gland was statistically significantly
decreased when compared to controls (approx. 35 % below controls). This difference was also statistical significant when related to brain weight
(approx. 35 % below controls), however not when related to body weight (approx. 20 % below controls).
 
Kidney weight was also statistically significantly increased in female animals of the HD group, when compared to controls (absolute weight approx.
25 %, when related to brain weight approx. 27 % and when related to body weight 21 % higher than controls). Kidney weight was also statistically
increased in male animals of this group when related to body weight (approx. 20 %). Higher kidney weight was not associated with any
histomorphological abnormalities.
In males of the HD group, absolute heart weight was slightly but statistically significantly lower (approx. 15 %) than in controls. However, as the
difference was not statistically significant, when heart weight was related to body or brain weight. Female animals were not affected and
histomorphologically no alteration was observed in the heart of male animals of the HD group. Thus, this finding is not assumed to be toxicologically relevant.
A slight but statistically significantly higher weight of adrenal glands (approx. 30 % higher than controls) of female animals of the LD group and in the HD group (only when related to brain weight) is not assumed to be toxicologically relevant as no dose-dependency was observed, it was not evident
in male animals and was not associated with histomorphological abnormalities.

Histopathology
Brain and Spinal Cord
Gliosis and/or neuronal cell loss with vacuolar change which was probably due to degeneration/collapse of nerve fiber projections, were recorded
symmetrically in the areas including olivary nucleus, cochlear nucleus, trigeminal nucleus, reticular nucleus, and/or locus coerules/mecencephalic
nucleus in both sexes of the HD group, and were recorded symmetrically or unsymmetrically in the area including thalamic nucleus in males of the HD group. The same lesions were also recorded in gray matter of the cervical spinal cord in two males of the HD group.
Gliosis and neuronal cell loss are the findings representing necrotic injuries in the central nervous system, which means that the test item produces
necrotic adverse events in the central nervous system.
Epididymides
Increased incidence and severity of spermatic granulomas were recorded in the HD group. Especially higher incidence and severity of granulomas
that occurred in both parts of corpus and cauda were noted in the HD group. In addition, diffuse interstitial edema with inflammatory cell infiltrate,
immature appearance of duct epithelium and caudal sperm stasis, except for male no. 20 in which there was cellular/tissue debris stasis, were
observed in all HD males, and occasional duct dilatation were also recorded in HD males. These were the lesions related to tissue destruction and/or
functional disturbances, and therefore, were considered adverse under the condition of this study.
Although the duct epithelium in all HD males appeared immature, there were no other indicators of cellular injury in the duct epithelium of any males.
In one male (no. 20) of the HD group, macroscopic small size of testes, epididymides, prostate, seminal vesicles and coagulating glands were
recorded, and diffuse degeneration/ atrophy in testes, oligospermia in epididymides, and reduced secretion in prostate, seminal vesicles and
coagulating glands were observed microscopically. However, the histologic alterations that could be attributed to treatment with the test item were
not observed in testes and the accessory sex glands in any other HD males despite the presence of treatment-related epididymal lesions, and
therefore, the above mentioned findings in no. 20 were considered to be of spontaneous lesions.
Liver
Centrilobular hepatocellular hypertrophy was recorded in both sexes of the HD group.
There were no further histomorphologic indicators of liver injury in any animals.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 2, 3 and 4 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 103.1%, 99.3% and 96.5% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose
groups, as mean of measured concentration did not differ from nominal concentration by more than 15%.
Stability of formulation samples was investigated in study week 1 for LD and HD groups. After 6 hours storage at room temperature recovery
compared to start value was 97.1% and 98.5% for LD and HD sample, respectively. All samples were stable, as concentration after storage did not
differ from start value by more than 15%.
Homogeneity of formulation samples was determined in study week 1 and 4 for LD and HD groups. The mean recovery observed for LD dose group
was 103.8% and 107.2% of the nominal value and 87.0% and 106.7% of the nominal value for HD group. The coefficients of variation of the different
sampling locations (top, middle, bottom) were 0.6% and 0.3% in LD dose group and 1.3% and 0.2% in HD group. All samples were homogenous, as COV was below or equal 15%.

Dose descriptor:

other: NOEL and NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

On the basis of this 28-Day Repeated Dose Oral Toxicity study with the test material in male and female Wistar rats with dose levels of
25, 75, and 200 mg/kg/d the following conclusions can be made:
The no-observed-effect level (NOEL) was established at 75 mg/kg/d.
The no-observed-adverse effect level (NOAEL) was established at 75 mg/kg/d in both sexes under the condition of this study.
The central nervous system and male reproductive system (epididymides) are considered to be the target organs of the test material.
Results from female animals also suggest the red blood system as a potential target system.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test material via oral administration to rats over a period of 28 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 28 days. Animals of an additional control group were handled identically as the dose groups but receivedthe vehicleDMSO and PEG400 (ratio 1:5) in this study. The 4 groups comprised of 5 male and 5 female Wistar rats.

During the period of administration, the animals were observed precisely each day for signs of toxicity. All animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly.

At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

A full histopathological evaluation of the tissues was performed on high dose and control animals.

In addition, histopathological evaluation ofliver, brain, spinal cord and epididymideswere perfomed for animals of low and medium dose groups as these were considered potential target organs. Any gross lesion macroscopically identified was examined microscopically in all animals.

The following doses were evaluated:

Control:                                    0                               mg/kg body weight

Low Dose:                    25       mg/kg body weight

Medium Dose:              75      mg/kg body weight

High Dose:                   200    mg/kg body weight

The test item formulation was prepared once weekly. Aliquots were prepared and stored between -15 and -35 °C. On each morning one aliquot was thawed and used for administration daily during a 28-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurements.

Summary Results

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

No test item related clinical signs of systemic toxicity were observed during the treatment period of this study.

Slight clinical symptoms (i.e. moving the bedding and salivation) is possibly partly associated to local reactions caused by the test item formulation during the procedure of oral application via gavage.

There were no ophthalmoscopic findings in any of the animals of this study.

Daily treatment with the test material had no effect on neurobehavioural parameters analyzed at the end of the treatment period.

Weight gain was slightly but significantly decreased in the high dose group when compared to controls, mainly during the last treatment week. At the end of the treatment period body weight of male – but not female dosed animals was between 8 and 10 % lower, when compared to controls. Even though a slight but statistically significantly decreased weight gain was also observed in males of the low dose (LD) group, the effect of daily oral treatment with the test material on body weight development of males is not assumed to be toxicologically relevant at the medium and low dose levels, as body weights were slightly lower (approx. 5 %) already on study day 1.

This correlated with slightly lower food consumption in the male animals of the high dose (HD) group. Food consumption of female animals was not affected in female animals of this group or at the low and medium dose levels.

Slightly lower levels of red blood cell parameters RBC, Hb and Hct were observed at the end of the treatment period at a dose of 200 mg/kg/d, when compared to controls - more prominently in female than in male animals. In female animals of this dose group reticulocytes were slightly elevated at the same time.

In 1/5 male animals of the HD group increased markers of hepatotoxicity (ALAT, ASAT) were associated with increased liver weight and centrilobular hepatocellular vacuolation as well as inflammatory cells in the liver. Besides, no toxicologically relevant effect on parameters of clinical biochemistry was observed at the end of the treatment period.

At the end of the treatment period there were no conspicuous findings in urinary parameters of treated animals.

At the end of the treatment period there were no macroscopic findings that could be related to the test material.

A slightly increased liver weight observed in female animals at a dose level of 200 mg/kg/d was associated with centrilobular hepatocellular hypertrophy.Thisfindingwasconsidered to be of adaptive character and not to be adverse under the condition of this study.

Moderately increased spleen weight in female animals at a dose level of 200 mg/kg/d is possibly related to a compensatory reaction against changes observed in red blood cell parameters. A histomorphological correlate, however, was not found.

Microscopically, gliosis and/or neuronal cell loss with vacuolar change which was probably due to degeneration/collapse of nerve fiber projections, were recorded symmetrically in the areas including olivary nucleus, cochlear nucleus, trigeminal nucleus, reticular nucleus, and/or locus coerules/mecencephalic nucleus in both sexes of the HD group (200 mg/kg/d), and were recorded symmetrically or unsymmetrically in the area including thalamic nucleus in males of the HD group (200 mg/kg/d). The same lesions were also recorded in gray matter of the cervical spinal cord in two males of the HD group.

Gliosis and neuronal cell loss are the findings representing necrotic injuries in the central nervous system, which means that the test item produces necrotic adverse events in the central nervous system.

Increased incidence and severity of epididymal spermatic granulomas were recorded in the HD group (200 mg/kg/d). Especially higher incidence and severity of granulomas that occurred in both parts of corpus and cauda were noted in the HD group. In addition, diffuse interstitial edema with inflammatory cell infiltrate, immature appearance of duct epithelium and caudal sperm stasis, except for male no. 20 in which there was cellular/tissue debris stasis, were observed in all HD males, and occasional duct dilatation were also recorded in HD males. These were the lesions related to tissue destruction and/or functional disturbances, and therefore, were considered adverse under the condition of this study.

Conclusion

On the basis of this 28-Day Repeated Dose Oral Toxicity study with the test material in male and femaleWistarrats with dose levels of 25, 75, and 200 mg/kgbody weight / day the following conclusions can be made:

The no-observed-effect level (NOEL) was established at 75 mg/kg/d.

The no-observed-adverse effect level (NOAEL) was established at 75 mg/kg/d in both sexes under the condition of this study.

The central nervous system and male reproductive system (epididymides) are considered to be the target organs of the test material. Results from female animals also suggest the red blood system as a potential target system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 28-Day Repeated Dose Oral Toxicity study was performed with the test material in male and female Wistar rats with dose levels of 25, 75, and 200 mg/kg/d. The following conclusions can be made:

The no-observed-effect level (NOEL) was established at 75 mg/kg/d.

The no-observed-adverse effect level (NOAEL) was established at 75 mg/kg/d in both sexes under the condition of this study.

The central nervous system and male reproductive system (epididymides) are considered to be the target organs of the test material. Results from female animals also suggest the red blood system as a potential target system.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only existing study

Repeated dose toxicity: via oral route - systemic effects (target organ) neurologic: central nervous system; urogenital: epididymides

Justification for classification or non-classification

Specific Target Organ Toxicity – Repeated Exposure: The notifiable substance did exhibit significant effects arising from a repeated exposure at a dose level of 200 mg/kg/day in male and female animals. As a result, the substance does meet the criteria for classification according to Regulation (EC) No 1272/2008, Annex I section 3.9. The substance is classified as STOT RE Cat 2, H373 May cause damage to organs (CNS and epididymides) through prolonged or repeated exposure.