Isobutylisopropyldimethoxysilane

Endpoint summary

Description of key information

The test substance was evaluated in a 28 day oral repeated dose toxicity study in the rat (OECD Guideline 407). Effects on the kidneys were observed in male rats, however, this was considered a species specific effect, which is not relevant to humans. The NOAEL for female rats was 1000 mg/kg bw/day.

An oral 90 day repeated dose toxicity study was conducted with the test substance in the rat (OECD Guideline 407) at the following doses: 5, 20, 100, 1000 mg/kg bw/day. Limited data are available regarding this study. The NOAEL for female rats was 100 mg/kg bw/day (however, the basis for selecting this dose as the NOAEL is not available). The NOAEL for male rats was 20 mg/kg bw/day based on adverse effects on the kidneys; this was considered a species specific effect, which is not relevant to humans.

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:

20 October 1987 to 20 November 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with OECD guidance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

other: Wistar- Han-Schering

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals and strain: 10 male and 10 female Wistar-Han-Schering rats per group/barrier sustained quality
Breeder: Schering AG
Acclimatization time: =/> 7 days
Caging conditions: Macrolon type II with wire-mesh bottom under conventional conditions
Room-no.: 0.526
Animals per cage: 1
Number of groups: 4
Body weight at start: 161-207 g for male and 155-178 g for female animals
Feed type: pulverized Altromin R
Feeding time: ad libitum 24 hours per day
Type of drinking water: tap water in water bottles
Watering time: ad libitum 24 hours per day
Room temperature: 21-22°C
Relative humidity: 48-68%
Light period: artificial; 12 hours dark/light rhythm (light 06.00 to 18.00 hours)
Randomization: by computer program
Identification: ear marks and corresponding cage label

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The animals were treated once daily in the morning intragastrically by gavage with ZK 119.649 as a solution in arachis oil at daily doses of 100, 300 and 1000 mg/kg body weight over a period of 28-31 days.

The following solutions of ZK 119.649 in arachis oil were prepared:
10 mg/ml
3 0 mg/ml
100 mg/ml

Fresh formulations were prepared daily during week 1 and weekly thereafter.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

28 to 31 days

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg body weight/day
Basis:
other:

No. of animals per sex per dose:

10 male + 10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

In the present study 1000 mg/kg were selected as the maximum dose based on the results in an acute oral toxicity study with the test substance in rats.

A control group with the same number and sex of animals received the same dose volume of the vehicle (10 ml/kg) in the same manner.

Positive control:

None

Observations and examinations performed and frequency:

Observations, clinical and laboratory studies
For laboratory studies blood samples were obtained from the jugular vein of each animal.
Furthermore no food was offered to the animals for more than approx. 16 hours prior to blood and urine sampling.

Mortality
A check for premature death and moribund animals was performed twice daily. The time of premature death was recorded.

General observations
All signs of weak health or reactions to treatment and behavioural changes were recorded. All animals were checked regularly during the application in the morning and thereafter in the afternoon. On weekends a second check was performed just before the technicians left the laboratory.

Food consumption
The amount of food consumed was recorded weekly. The mean food consumption per day between day 1 and 28 was evaluated statistically.

Water consumption
The quantity of water consumed by the individual animal was recorded twice weekly. The mean consumption of drinking water per day between day 1 and 29 was evaluated statistically.

Body weight
Body weight was recorded weekly. The weight gain from day 1 to 28 was calculated.

Ophthalmoscopy
Ophthalmoscopic examinations were carried out on day 29 in 5M and 5F animals per group. The pupils of all animals were dilated using a mydriaticum (Mydriaticum-Roche (R), 0.5%) and the eyes examined with the naked eye and an ophthalmoscope (Oculus-Visuskop, W. Oculus).

Hematology
Hematological investigations were performed on day 25 for determination of the following parameters:
Red cell and total white cell count, hemoglobin, hematocrit (packed cell volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration), MCV (mean corpuscular volume), reticulocyte count, thrombocyte count and differential count in 5M and 5F animals per group.

Bone marrow
At terminal sacrifice bone marrow was sampled from the femur of 5 male and 5 female animals of all groups. The number of nucleated cells/mg bone marrow was determined and a myelogram prepared. Only the marrow smears of groups 1 and 4 were examined.

Urinalysis
pH, specific gravity, protein, glucose, acetone, blood, urobilinogen, bilirubin and sediment of urine were determined on days 15 and 22-29 in 5M and 5F animals per group. The studies on day 15 were amended to the original study protocol, since a reddish-wet staining of the fur in the inguinal region had been observed in some animals of the mid and high dose group. Two male animals (no. 48, group 3 and no. 67, group 4) with this finding were included in these studies in addition to the above-mentioned 5M/5F.

Biochemistry
The following parameters were determined in serum from 5M and 5F animals per group: glutamic pyruvic transaminase (GPT), alkaline phosphatase (AP) on day 2 (approx. 22h after the first treatment) and on day 25; glucose, urea nitrogen, total cholesterol, total protein, protein electrophoresis, sodium, potassium, calcium and chloride on day 25.

Blood coagulation
The following parameters were determined in plasma from 3-5 male and female rats per group: thromboplastin time, activated partial thromboplastin time, thrombin time and fibrinogen on day 24.

Sacrifice and pathology:

On completion of treatment all animals were sacrificed under general chloroform anaesthesia. Each animal was rapidly exsanguinated by incision of the left or right jugular blood vessels. Immediately after occurrence of death, a full post mortem examination was performed and all macroscopic findings were noted.

Organ weights
At autopsy the following organs were dissected free of fat, removed and weighed, the paired organs being weighed together: liver, kidneys, heart, adrenal gland, thyroid, spleen, iliac lymph node, thymus, brain, pituitary, ovary, uterus, semical vesicle, testis, prostate, pancreas.

Microscopic examination
From all control and all treated animals the following organs/tissues or representative samples thereof were fixed in Lillie's buffered formalin (F) or in Bouin's fluid (B):

liver (F), (B), kidney (F), (B), heart (F), (B), adrenal gland (F), (B), thyroid with parathyroid gland (F), (B), lung (F), spleen (F), thymus (F), iliac lymph node (F), brain [cerebrum, cerebellum, medulla oblongata] (F), spinal cord, cervical (F), peripheral nerve [n. saphenus] (F), pituitary gland (B), ovary (F), uterus [horn] (F), mammary gland (F), testis (F), Harderian gland (F), prostate (F), seminal vesicle (F), stomach (F), duodenum (F), jejunum (F), ileum (F), cecum (F), colon (F), rectum (F), pancreas (B), salivary gland [gl. submandibulars] (F), vena cava caudalis (F), aorta thoracalis (F), trachea (F), esophagus (F), urinary bladder (F), skin (F), muscle [m. gastrocnemius] (F), eye (F), bone [femur with bone marrow] (F)

and any other tissue showing macroscopic alterations, if necessary for making a diagnosis.

Tissue samples from liver, heart, adrenal gland, thyroid and bone fixed in formalin were not processed for histological examination. For histological examination paraffin sections from all control and all high dose group animals were prepared and stained with hematoxylin and eosin (H.E.). In addition to the routine microscopic examination of Bouin fixed kidney samples of the animals from the low and high dose group and those showing macroscopic kidney changes, formalin fixed samples from both kidneys of all animals from all groups were examined histologically. For these special studies the formalin fixed specimens were chosen, because after fixation the macroscopic finding {grey-white foci) was only visible in these samples and not in Bouin-fixed material.

From the pancreas of all animals of the control and high dose group a second tissue sample was processed and examined histologically. Additionally, pancreas samples of the low and mid dose group animals were also processed, stained with H.E. and examined histologically, too.

Other examinations:

None

Statistics:

The Dunnett-test was used to assess the statistical significance of differences between group 1 (control) and the treatment groups.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Mortality:

mortality observed, treatment-related

Description (incidence):

See below for details

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See below for details

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY

No animal died during the course of the study.

Slight to marked sialorrhea after treatment was observed in both sexes at all three dose levels. This finding occurred for the first time in the high dose group (1000 mg/kg) on day 8 and was noted until the end of the experiment. It was dose-dependent with respect to incidence as well as severity. At the high dose this finding was also observed sporadically before treatment in some animals, but at a markedly lower incidence. Moreover, at doses of 300 mg/kg and 1000 mg/kg an increased occurrence of wet fur in the inguinal region in both sexes and of ruffled fur in male animals was noted. In two males of the mid dose group (no. 41 and 48) and in four males of the high dose group (no. 63, 65, 66 and 67) the inguinal region showed a red staining. Single male rats from the mid (no. 41 and 46) and high dose group (no. 67) revealed a transient slight apathy. Alopecia, thinning of fur and scab formation were noted only sporadically in single animals and not considered compound related.

BODY WEIGHT AND WEIGHT GAIN

A tendency towards a slightly reduced body weight gain was noted in the male animals of the mid and high dose group when compared to the control animals (statistically not significant). A box plot of the weight gain for the male rats shows that there was a relatively large scatter of the individual values, which made the assessment of a possible compound effect rather difficult.

In female animals no compound related effect on weight gain was observed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

No compound related effect on the intake of food was observed.

FOOD EFFICIENCY

Not evaluated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

The consumption of drinking water did not show a compound related effect.

OPHTHALMOSCOPIC EXAMINATION

No compound related findings were observed. Changes such as vesicular inclusions in the cornea or well-defined nucleus of the lens were not regarded as compound related because they were also observed in the control group with similar frequency or were sporadic alterations, respectively.

HAEMATOLOGY

No compound related hematologic changes were observed. The statistically significant changes in haemoglobin, erythrocyte count and haematocrit were not considered effects of treatment with ZK 119.649, because they did not show a dose-dependency.

BONE MARROW

No compound related effects on the bone marrow were observed. In animal no. 72F of the high dose group a decrease in the number of nucleated cells/mg marrow was noted due to a decrease in neutrophil granulocytopoiesis as well as a slight decrease in erythropoiesis. Since these alterations occurred in one animal only and no respective alterations were present in the peripheral blood, they were considered chance findings. The decrease in bone marrow lymphocytes in the female rats of the high dose group (p < 0.05) is not regarded compound related, since the values were within the reference range of this laboratory for the respective parameter.

CLINICAL CHEMISTRY

A slight increase in total serum cholesterol was observed in female rats of the mid (p < 0.05) and high dose group (p < 0.01) on day 25. At the same investigations a slight decrease in serum chloride was noted in the male rats from these two groups (p < 0.01) . All other statistically significant differences (p < 0.05 to p < 0.01) to the control are not considered as being of biological relevance either because of a lack of dose dependency or because the values remained within the reference range for these parameters. Moreover decreased serum activities or concentrations of GPT and urea nitrogen are not considered to be indicative of a pathological condition.

No compound related effects were observed in the coagulation studies. The statistically significant decrease (p < 0.05) in activated partial thromboplastin time in male rats of group 2 was not considered a compound related effect, because there was no such change at the two higher dose levels.

URINALYSIS

A slight increase in the specific gravity of the urine was noted on days 15 and 22-29 at the high dose of 1000 mg/kg in male (p < 0.01 to p < 0.05) and female rats (number of animals too small for statistical testing). Furthermore, an increase in urine volume was observed in the female animals of the high dose group. (not tested statistically, as above) on days 22-29. At both determinations the male animals of the mid and high group showed a
slight to moderate increase in the concentration of protein and blood in urine, while hematuria was also observed in three of five male rats of the low dose group on days 22-29. This finding may serve as an explanation for the above-mentioned reddish staining of the fur in the inguinal region in some rats. (However, the occurrence of blood in animal no. 48M of group 3 was rated only "slight" on day 15 and "traces" on days 22-29, although the red stain of fur had been noted in this rat.)

In addition, at the mid and high dose level the male rats had a slight increase in the number of epithelial cells in the urine samples on days 22-29. Aggregates of cell detritus were observed in individual male animals of the mid and high dose group on day 15 and in all male rats from these two groups on days 22-29. The same finding occurred in two male rats (no. 22 and 23) of the low dose group on days 22-29. The aggregates showed an appearance like disaggregated granulated cylinders. Since this finding was noted neither in dosed female animals nor in male control animals, it seems to be a sex-specific, compound related effect.

NEUROBEHAVIOUR

Nor evaluated.

ORGAN WEIGHTS

The organ weights which showed statistically significant differences from the corresponding values in control animals. The analysis of absolute and relative organ weights did not reveal any clear-cut compound related effect. The slight increase (p < 0.01) in absolute and relative liver weights in females of the high dose group is not considered to be biologically relevant, because the absolute values lie just at or slightly above the upper borderline of the reference values (90% range) for historical control animals of the same body weight range, whereas the mean values of the relative liver weights are in accordance with corresponding reference values. This evaluation is also supported by the results of the histological examination of the corresponding liver samples, which did not reveal any morphological finding which could explain an increase in weight.

The slight increase in kidney weights in female animals of the mid and high dose group and in the males of the high dose group is also not considered to be of biological relevance, because the mean values of these groups are in accordance with those of corresponding reference values. The statistical significance is presumably due to the low values of the control animals (when compared to historical controls). This is also true for the increase in the weights of the uterus observed in the high dose group which however showed a large scatter of the uterus weight in individual animals.

The statistically significant increase of relative adrenal weights in male animals of the high dose group and of absolute and relative thymus weights in females of the low dose group as well as the decrease in absolute weights of the testes of the animals of the mid dose group are considered to be
chance events, because there was a lack of dose dependence and/or the values match to the corresponding reference values. These organ weight changes are therefore not attributed to effects of the compound under test.

GROSS PATHOLOGY

The occurrence of grey-white punctiform foci on the surface of both kidneys in male animals of all groups treated with ZK 119.649 is attributed to administration of the test compound. For this finding, however, no histological correlate was found. In addition the paleness of kidneys observed in two male rats of the high dose group is suspected to represent a compound related effect.

All other findings observed were either also present in control animals (focal reddening in thymus, dilatation of renal pelvis) or are spontaneous findings which are occasionally seen in rats of the rat strain used in this study (hydrometra, cystic dilatation of ovarian sac, gray-white covering of gastric mucosa). The subcutaneous hematoma in the cervical region is considered to be a sequela of the blood sampling from the jugular vein.

HISTOPATHOLOGY: NON-NEOPLASTIC

The only finding noted at histological examination which could be interpreted to be influenced by the treatment with the test compound was an increase in the incidence and severity of a lesion called progressive nephropathy in male animals of the medium and high dose group.

The lesion was characterised by basophilia of cytoplasm of tubular epithelial cells and hypertrophy of nuclei and thickening of basement membrane, partly associated with scattered interstitial accumulations of mononuclear cells. Partly the lesion consisted of cytoplasmatic basophilia, very slightly hypertrophied nuclei and slightly flattened epithelium accompanied by slight dilatation of tubular lumen without any remarkable thickening of basement membrane. In some male animals mainly in the high dose group also proteinaceous masses were observed in the tubular lumen. In these cases the lesion was designated as moderate. In all cases only few nephrons were concerned.

All other lesions observed were not attributed to effects of the test compound, because they were either also present in the control animals and/or are known to occur spontaneously in rats with the same frequency and intensity (severity).

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: In male rats, there was an increase in the occurrence of blood and aggregates of cell detritus in the urine and grey-white punctiform foci on the surface of the kidneys.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

When ZK 119 .649 was given orally by gavage to rats over a period of 28-31 days, sialorrhea in both sexes as well as hematuria, aggregates of cell detritus in the urine and grey-white punctiform foci on the surface of the kidneys in male animals were observed at the lowest dose of 100 mg/kg. Although microscopic examination of these animals did not reveal any particular morphological changes in comparison to the control animals, the findings in male rats are suggestive of an early stage of kidney damage. This presumption is supported by the increased incidence of progressive nephropathy which was noted in male rats at dose levels of 300 and 1000 mg/kg. Since this lesion is known to occur spontaneously in laboratory rats, the administration of ZK 119.649 appears to have acted as an accelerating factor. In the female rats of the high dose group there was an increase in the specific gravity and the volume of urine, but no evidence of organ damage was noted in the females of all three groups.

A NOAEL could not be determined for males as treatment-related adverse effects occurred at the lowest dose tested (100 mg/kg bw/day). The LOAEL for males is 100 mg/kg bw/day. The NOAEL for female rats is 1000 mg/kg bw/day. Sialorrhea was observed in female rats at dose

Executive summary:

For the toxicological evaluation of ZK 119.649, a new alkyl silane, the systemic toxicity in rats was to be studied after daily oral (intragastric) administration over a period of 28-31 days. The experiment was to be conducted in accordance with the OECD Guidelines for Testing of Chemicals (no. 407, adopted May 12, 1981).

10 male and 10 female Wistar-Han Schering rats per group received ZK 119.649 as a solution in arachis oil orally by gavage at daily,

doses of 100, 300 and 1000 mg/kg body weight over a period of 28-31 days. A control group with the same number and sex of animals received the same dose volume of the vehicle (10 ml/kg) in the same manner.

General observations (daily) and measurements of body weight, food and water consumption (once/twice weekly) were performed in

all animals. Ophthalmoscopic examination was conducted on 5 male and 5 female animals per group on day 29. Hematology, urinalysis, biochemistry and coagulation studies were performed on 5 animals per sex and group in week 4-5, serum enzyme determinations (GPT, AP) also on day 2. Additional urinalyses were conducted on day 15. At terminal sacrifice all animals were subjected to a full necropsy and selected organs were weighed. Subsequently bone marrow and histological examinations were performed. The Dunnett-test was used for statistical analysis.

The following changes are considered to represent effects of ZK 119.649:

At a dose of 100 mg/kg and higher:

Dose-dependent slight to marked sialorrhea after treatment.

Increase in the occurrence of blood and aggregates of cell detritus (appearing like disaggregated granulated cylinders) in the urine of male rats on days 22-29 (at 100 mg/kg) or on days 15 and 22-29 (at higher dose levels), respectively.

Grey-white punctiform foci on the surface of the kidneys in male animals (not confirmed histologically).

At a dose of 300 mg/kg and higher:

Increased incidence of wet fur in the inguinal region in both sexes and of ruffled fur in male rats only.

Red stain of the fur in the inguinal region of male animals.

Transient slight apathy in individual male animals.

Tendency to a slight reduction of body weight gain in male animals (statistically not significant).

Slight to moderate increase in the occurrence of protein and blood in the urine of male rats on days 15 and 22-29.

Slight increase in the number of epithelial cells in the urine of male animals on days 22-29.

Slight increase in total serum cholesterol in female rats on day 25 (p < 0.05 to p < 0.01).

Slight decrease in serum chloride in male rats on day 25 (p < 0.01).

Increase in the incidence and severity of an early stage of progressive tubular nephropathy in male rats characterised by hypertrophy of nuclei, basophilia of tubular epithelial cells and thickening of basement membrane.

Additionally at a dose of 1000 mg/kg:

Sporadic slight sialorrhea before treatment.

Slight increase in the specific gravity of the urine in male (p < 0.01 and p < 0.05) and female rats (numbers of animals

too small for statistical testing) on days 15 as well as 22-29.

Increase in the urine volume of female animals on days 22-29 (number of samples too small for statistics).

Paleness of the kidney in two male animals.

When ZK 119.649 was given orally by gavage to rats over a period of 28-31 days, sialorrhea in both sexes as well as hematuria, aggregates of cell detritus in the urine and grey-white punctiform foci on the surface of the kidneys in male animals were observed at the lowest dose of 100 mg/kg. Although microscopic examination of these animals did not reveal any particular morphological changes in comparison to the control animals, the findings in male rats are suggestive of an early stage of kidney damage. This presumption is supported by the increased incidence of progressive nephropathy which was noted in male rats at dose levels of 300 and 1000 mg/kg. Since this lesion is known to occur spontaneously in laboratory rats, the administration of ZK 119.649 appears to have acted as an accelerating factor. In the female rats of the high dose group there was an increase in the specific gravity and the volume of urine, but no evidence of organ damage was noted in the females of all three groups.

A NOAEL could not be determined for males as treatment-related adverse effects occurred at the lowest dose tested (100 mg/kg bw/day). The LOAEL for males is 100 mg/kg bw/day. The NOAEL for female rats is 1000 mg/kg bw/day. Sialorrhea was observed in female rats at dose </= 1000 mg/kg, however, this is considered a physiological response rather than a sign of systemic toxicity. An increase in volume and specific gravity of the urine was noted in female rats in 1000 mg/kg group, however, no statistical analysis was possible and there was no evidence of organ damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted in accordance with OECD Guideline therefore rated as Klimisch 1 study.

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

When the test substance was administered orally by gavage to rats over a period of 28-31 days, sialorrhea was observed in both sexes. In addition, in male animals, hematuria, aggregates of cell detritus in the urine and grey-white punctiform foci on the surface of the kidneys in male animals were observed at the lowest dose of 100 mg/kg. Although microscopic examination of these animals did not reveal any particular morphological changes in comparison to the control animals, the findings in male rats are suggestive of an early stage of kidney damage. This presumption is supported by the increased incidence of progressive nephropathy which was noted in male rats at dose levels of 300 and 1000 mg/kg. Since this lesion is known to occur spontaneously in laboratory rats, the administration of ZK 119.649 appears to have acted as an accelerating factor. In the female rats of the high dose group there was an increase in the specific gravity and the volume of urine, but no evidence of organ damage was noted in the females of all three groups. A NOAEL could not be determined for males as treatment-related adverse effects occurred at the lowest dose tested (100 mg/kg bw/day). The LOAEL for males is 100 mg/kg bw/day. The NOAEL for female rats is 1000 mg/kg bw/day. Sialorrhea was observed in female rats at dose </= 1000 mg/kg, however, this is considered a physiological response rather than a sign of systemic toxicity. An increase in volume and specific gravity of the urine was noted in female rats in 1000 mg/kg group, however, no statistical analysis was possible and there was no evidence of organ damage.

In the 90 day oral repeated dose toxicity study, nephrotoxicity occurred in male rats administered doses of >/= 100 mg/kg bw/day. When considering the conclusions to be drawn for man, the toxicological relevance of the entirety of these findings does not appear to be of immediate relevance since it could be shown (10 day comparative study with the test substance and 2,2,4 trimethylpentane, expert report by G.H. Pigott) that sex and species-specific findings in the kidney are due to a particular reaction (alpha 2 microglobulin) and this mechanism is not present in other species including man. This is supported by the results of the reproductive/development screening test with the test substance, which showed hyaline drops in the kidneys in male rats. The hyaline droplets likely represented alpha2 microglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation, leading ultimately to proximal cortical tubule cell injury, the formation of granular casts and increased cell turnover as manifested by tubular basophilia. The combination of renal lesions observed at 300 and 1000 mg/kg has been referred to as male rat progressive nephropathy. This effect on the kidneys was not observed in female rats. This protein (alpha2 microglobulin) is not present in female rats or in higher mammals, including man, and thus has little relevance to the human condition.

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

This study has been selected because it is a well documented GLP study conducted in accordance with an EU Guideline. A 90 day study is available, however, only limited data are available for this study therefore it has not been selected. The NOAEL value for the female rats is selected; effects on the kidneys were observed in male rats, however, this was considered a species specific effect, which is not relevant to humans.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

The oral route is considered the most appropriate route.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

The oral route is considered the most appropriate route.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:

The oral route is considered the most appropriate route.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:

The oral route is considered the most appropriate route.

Justification for classification or non-classification

The above studies have been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the study was conducted to GLP and in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. The nephrotoxicity observed in male rats is considered to be a species specific effect, which is not relevant to humans, therefore the above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).