1,3-Benzenedimethanamine, reaction products with polyethylene glycol mono-Bu ether and TDI

Endpoint summary

Description of key information

 In a Combined Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with WS400517 in rats (OECD 422) at doses of 100, 300, and 1000 mg/kg bw/day with dosing for five consecutive weeks the NOEL was derived at 1000 mg/kg bw/day.
 In a sub-chronic oral (gavage) toxicity study the highest dose of WS400517 that practically could be administered to the animals was 600 mg/kg bw/day. This level was derived as NOAEL.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

of 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sprague Dawley rats, strain: Crl:CD(SD) with appropriate range of bodyweight at study start.
- Source: Charles River (UK) Ltd.
- Age at treatment start: 71 days.
- Weight at treatment start: Males: minimum 346 g, maximum 392 g,
Females: minimum 226 g, maximum 273 g.
- Housing Inside a barriered rodent facility:
all animals pre-pairing + toxicity subgroups: In groups up to 5 by sex in solid floor polycarbonate cages.
during pairing (1 male+1 female/cage): In RB3 modified polypropylene cages with stainless steel grid-floor over absorbent paper-lined trays.
males after pairing: In groups up to 5 in solid floor polycarbonate cages.
females during gestation and lactation: Females housed individually (+litter) in solid floor polycarbonate cages.
- Bedding material (in solid floor cages): Wood based bedding, sterilised by autoclaving before use.
- Cage enrichment: Aspen chew block + plastic shelter (except during pairing or post gestation day 20).
- Diet (ad libitum): Standard rodent diet (SDS VRF1 Certified) without antibiotic, chemotherapeutic or prophylactic agent.
- Fasting (diet withheld): Main phase males and Toxicity phase females overnight before blood sampling for clinical pathology.
- Water (ad libitum): Potable drinking water from the public supply.
- Acclimation period: 6 days before treatment start, under laboratory conditions.

Routine analysis of the batch of diet used and water, chew blocks and bedding material did not provide evidence of contamination that might have prejudiced the study.

IN-LIFE DATES:
- Duration of test, males & toxicity phase females: Five weeks
Duration of test, main phase females (i.e. reproductive subgroup): From 14 days prior to pairing to day 7 of lactation.
Duration of test, offspring: From birth to day 7 of lactation.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
- Rate of air exchange: At least 15 changes/h
Deviations from the target ranges for temperature and relative humidity were not evident.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

- Concentration in vehicle: The concentration of the test material in vehicle varied between dose groups thus allowing constant dosage volume in terms of mL/kg bw/day.
- Amount (dose volume by gavage): 5 mL/kg bw/day..
Actual dose volumes were calculated at about weekly or shorter intervals accounting for the latest body weight. Litter animals were not dosed.

- For concentrations of test material in vehicle at different dose levels, see Table 1 in "Any other information on materials and methods incl. tables"

- Justification for choice of vehicle:
The suitability of propylene glycol as a vehicle was established during the 7-day range-finding study:
Endpoint study record "7.5.1 Repeated dose toxicity: oral - 7d_range-finding_gavage_HLS_GAH0058".
In addition, in the present main study, concentrations of dose formulations were chemically analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Chemical analysis of test material formulations by high performance liquid chromatography coupled with a mass spectrometer (HPLC-MS/MS).
- Concentrations (verified at first and last treatment week) of the test material formulations were confirmed at each dose level.
- Chemical analysis confirmed that the mean concentrations of WS400517 in prepared formulations were 95.5% to 101.5% of the corresponding
nominal concentration, thus confirming accuracy of formulation.

Duration of treatment / exposure:

- Treatment period, males & toxicity phase females: Daily, for five consecutive weeks, in males commencing 14 days prior to pairing
- Treatment period, main phase females (i.e. reproductive subgroup): 44 to 57 days (from 14 days prior to pairing to day 6 of lactation)
- Offspring were not dosed

Frequency of treatment:

Daily, 7 days/week (during parturition, dosing omitted as appropriate)

Remarks:

Doses / Concentrations:
0 (vehicle control), 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Toxicity phase animals: */ 5 females
Main phase animals (i.e. reproductive subgroups): 10 males / 10 females
*Explanatory note by the notifier:
Examinations assigned to the toxicity phase females to meet the requirements of a 28-day repeat dose oral toxicity study were also assigned to 5 (for some examinations to 10) main phase males per dose group. Therefore, these 5 main phase males per dose group are called also "toxicity subgroup" in the present robust study summary for clarification. After pairing with main phase females, all male survivors were killed at the same time (Week 6).

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, animals initially entering the study were divided into toxicity subgroup animals (toxicity phase) and reproductive subgroup animals (main phase), whereby 5 of the 10 F0 males (used for pairing) per dose group formed the toxicity male subgroups.

Dose selection was based on the results of a 7-day preliminary oral toxicity study in the rat in which dose levels of 100, 300 or 1000 mg/kg/day did not have any overt treatment-related effects on young adult animals (females nulliparous and non-pregnant).

Positive control:

Not included in the study.

Observations and examinations performed and frequency:

Clinical observations performed and frequency:
- Clinical signs : At least twice a day (before and after administration)
- Detailed physical examination
and arena observations: Before treatment start and at least once a treatment week.
- Functional Observation Battery:* During treatment week 5 (before dosing) on all toxicity subgroup animals (5 males + 5 females/group).
- Body weight, all males: Weekly throughout the study.
Body weight, Toxicity Females: Weekly throughout the study.
Body weight, Repro. Females: Weekly for pre-pairing period; on gestation days 0, 6, 13, 20; on lactation days 1 & 7.
- Food consumption, all males: Weekly for pre-pairing period and for the period after mating.
Food cons., Toxicity Females: About weekly throughout the study.
Food cons., Repro. Females: Weekly for pre-pairing period, during gestation for days 0-6, 6-13, 13-20, during lactation for days 1-4 & 4-7.

* FOB including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength and motor activity.

Hematological examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Red blood cell count, reticulocyte count, white blood cell count, platelet count, hemoglobin concentration, hematocrit value, differential leukocyte counts,
protrombin time, activated partial thromboplastin time, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration.

Blood (plasma) chemical examinations (only for toxicity subgroup animals) during treatment week 5 after functional observation battery:
Total protein, albumin, A/G ratio, urea, creatinine, glucose, total cholesterol, total bilirubin, bile acids, sodium, potassium, chloride,
calcium, inorganic phosphorus, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase.

This study was conducted to examine both repeated dose toxicity and  reproductive/developmental toxicity as an OECD screening combined study
(OECD 422 test guideline).  Therefore, some of the examinations were confined to toxicity subgroup animals, as indicated above.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

Terminal sacrifice
- all males and toxicity subgr. females: Killed in Week 6, after completion of the Treatment Week 5 investigations.
(1 mid dose male was found dead on the day of scheduled termination)
- reproductive subgr. females & offspring: Killed on Day 7 post partum.
(1 low dose female was killed following the death of her litter on lactation day 1)

Gross pathology:
- adult/parental animals: Full macroscopic examination with tissue collection.
- offspring: Full macroscopic examination including assessment of the presence of milk in the stomach, where possible.
(Missing or grossly autolysed or cannibalised offspring could not be examined).

Organs Weights:
- main phase and tox. subgr. adults: Adrenals, brain, epididymides, heart, kidneys, liver, lungs & bronchi, ovaries, pituitary, prostate, seminal vesicles
& coagulation gland, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix & oviducts.

Histopathology:
- toxicity subgroups: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, eyes, Harderian glands, optic nerves, pituitary gland, thyroid with parathyroids, heart, thymus, liver, spleen,
adrenals, kidneys, testes, epididymides, ovaries, lung, trachea, esophagus, stomach, duodenum, jejunum, ileum,
caecum, rectum, colon, Peyer's patch, lymph node (axillary, mesenteric), urinary bladder, uterus (with cervix &
oviducts), vagina, spinal cord, sciatic nerve, skeletal muscle, skin with mammary glands, sternum with marrow,
seminal vesicle & coagulation gland, prostate. In addition, any gross lesions for all adult animals from all dose groups
were examined by light microscopy.
- reproductive subgroups All above organs/tissues from premature deaths (1 low dose female and 1 mid dose male) and any gross lesions from all adult animals from all dose groups were examined by light microscopy.

Other examinations:

Reproductive and developmental toxicity parameters (addressed in separate endpoints).

Statistics:

Grip strength, motor activity, bodyweight, food consumption, organ weight, litter size, survival indices & clinical pathology data were statistically analysed by adoption of the following sequence of tests:

- Parametric analysis, if Bartlett's test for variance homogeneity was not significant at the 1% level.
F1 approximate test for monotonicity of dose-response. If this F1 test was not significant at the 1% level, Williams' test for a monotonic trend was applied.
If this F1 test was significant, suggesting that the dose-response was not monotone , the Dunnett's test was performed instead.

- Non-parametric analysis, if Bartlett's test was still significant at the 1% level following logarithmic and square-root transformations.
H1 approximate test for monotonicity of dose-response. If this H1 test was not significant at the 1% level, Shirley's test for a monotonic trend was applied.
If this H1 test was significant, suggesting that the dose-response was not monotone, the Steel's test was performed instead.

-For grip strength, motor activity, survival indices and clinical pathology data,
if 75% of the data (across all groups) were the same value, pairwise comparison of each dose group against the control by Fisher’s Exact tests.

-For organ weight data, covariance analysis using terminal bodyweight as covariate (Angervall & Carlstrom, 1963).

Sex ratio
- Analysis by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991).
Each treated group was compared to control using a Wald chi-square test.

For statistical references, see next field.

Clinical signs:

no effects observed

Description (incidence and severity):

attributable to treatment with the test material

Mortality:

no mortality observed

Description (incidence):

attributable to treatment with the test material

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery including sensory reactivity tests (approach, touch, auditory startle reflex, tail pinching), grip strength, motor activity and attention to clinical signs of neurotoxicity during animal observations.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS, NEUROBEHAVIOUR AND MORTALITY
One female of the low dose group (100 mg/kg/day) was killed following the death of her litter on lactation day 1 and one male of the mid dose group (300 mg/kg/day) was found dead on the day of scheduled termination. These deaths were not attributable to treatment with WS400517 and their causes could not be elucidated. In the mammary glands of the affected dam only slight secretory activity was evident.

Toxicologically relevant clinical signs or effects on sensory reactivity, grip strength or motor activity were not evident. Transient chin rubbing occurring more frequently with increasing dose and isolated incidences of salivation in a few animals at all dose levels may have been related to palatability of the dose formulations rather than being indicative of toxicity.

BODYWEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Bodyweight and food consumption were unaffected by treatment with WS400517 in treated males and nulliparous, nonpregnant females. In main phase females receiving 300 or 1000 mg/kg bw/day, mean bodyweight gain was slightly higher than concurrent control during gestation Days 0-6 and mean food consumption slightly higher during gestation days 0-13. These minor differences from concurrent controls were not considered to represent an adverse effect.

CLINICAL PATHOLOGY
Toxicologically significant changes in haematology or clinical chemistry (blood plasma) parameters were not evident.

ORGAN WEIGHTS
Toxicologically significant effects on organ weights were not evident in the present study.

GROSS PATHOLOGY AND HISTOPATHOLOGY (NON-NEOPLASTIC)
Macroscopic or microscopic pathology findings attributable to treatment with the test material were not evident.

OTHER RESULTS
Reproductive and developmental toxicity parameters are addressed in separate endpoints.

Key result

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2016 to June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BYK Chemie HJ-15-097
- Expiration date of the lot/batch: 1-Dec-2016
- Purity test date: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: grinding

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK)
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 44 - 50 days
- Weight at study initiation: males 213-276 g, females 158-201 g
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: Teklad 2014C diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed. Small amounts of the vehicle were added and mixed with the test material in a mortar using a pestle. Any agglomerates were broken down to produce a smooth paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was poured into a measuring cylinder which had been wetted with the vehicle. The mortar was also rinsed with the vehicle and added to the cylinder and the suspension was made up to the required volume with vehicle. The suspension was transferred into a mixing container and mixed, using a Silverson mixer, carefully until a slight bitty suspension was achieved (not a paste) and then stirred magnetically for at least 30 minutes.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 20, 60, 120 mg/ml
- Amount of vehicle (if gavage): 5 ml / kg bw
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of dose formulations were analysed by HPLC with evaporative light scattering detector. Mean concentrations were within 10% of the nominal concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

The highest dose should have been at 1000 mg/kg bw/day but the dose formulation at this concentration turned out to be unable to be administered because the formulation could not be drawn up the catheter.

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: In an OECD422 study in the CD rat, oral administration of WS400517 (BYK-LP R 6075 WS) at dose levels of 100, 300 and 1000 mg/kg/day was well tolerated with no adverse effect of treatment on clinical signs, body weight gain, macroscopic finding or organ weight. It was concluded from these studies that the high dose of 1000 mg/kg/day was the no-observed-effect-level (NOEL).

Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (equivalent to the limit dose for this study type) on the current study. The intermediate and low dose levels of 300 and 100 mg/kg/day were selected to assess any dose-responsiveness of any test substance-related finding following a longer (i.e. 13 week) treatment period.

On Day 1 of study, however, the 1000 mg/kg/day dose level was unable to be administered because the formulation could not be drawn up the catheter (8 or 9 choke). Reducing the concentration by increasing the dose volume was discounted because of the toxicity of larger amounts of the vehicle, propylene glycol. The highest practical dose, considered to be 600 mg/kg/day (120 mg/ml administered at 5 ml/kg) was, therefore, administered for the high dose group from Day 2.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once per week starting 1 week before first adminstration

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in week 12
- Dose groups that were examined: all animals (pre-treatment), animals of high dose group and control group (week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly arena observations
- Dose groups that were examined: all animals of all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: week 12, all animals

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Estrous cycles by vaginal smear: for 14 days during weeks 10 and 11 of treatment, all groups

Sperm analysis: after scheduled sacrifice of each male, sperm motility (all groups), morphology and homogenisation-resistant spermatid count (groups 1 and 4)

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, body weight, sperm analysis, organ weight and clinical pathology data: A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see "overall remarks, attachments" : Figure body weight

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological investigations during Week 13 of treatment revealed slightly high reticulocyte counts, compared with controls (1.4X control), for males receiving 600 mg/kg/day. Activated partial thromboplastin time was slightly shorter than that of the controls for females receiving 300 or 600 mg/kg/day and the mean prothrombin time was shorter than that of the controls for females receiving 600 mg/kg/day.
A small decrease of mean cell haemoglobin concentration at all doses in males attained statistical significance, however, the differences were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation.
see "overall remarks, attachments" : Table Haematology group mean values

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations during Week 13 of treatment revealed slightly low (0.88X control) urea concentration for females receiving 600 mg/kg/day.
A small number of other differences from controls attained statistical significance, but these were minor, confined to one sex and lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included a reduction in bile acids for males at 300 or 600 mg/kg/day and a slight increase in total protein in males at 600 mg/kg/day.
see "overall remarks, attachments" : Table Clinical biochemistry group mean values

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Analysis of organ weights for animals killed after 13 weeks of treatment revealed, when compared with the controls, slightly high absolute and body weight adjusted spleen weights for males receiving 600 mg/kg/day. Following microscopic examination of the spleen, no findings were seen to account for the reported slightly increased spleen weights for males that received 600 mg/kg/day.

Absolute and body weight-adjusted kidney weights at all doses in females were slightly lower than those of the controls. The differences from controls were, however, minor, there was no dose relationship and no similar trend in the kidney weights of males and, consequently, these differences from controls were considered of no toxicological significance.

All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
see "overall remarks, attachments" : Table Organ weights

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with BYK-LP R 6075 WS were seen in the mandibular salivary gland of females. A minimal to moderate decrease in the granules in the striated ducts was seen in treated females and exhibited a dose-relationship. The toxicological significance of this finding in one of the three salivary glands was unknown.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

All other findings were considered incidental and unrelated to treatment, attributed to normal biological variation.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: NOAEL refers to the highest dose level that practically could be administered to the animals.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

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

Justification for classification or non-classification

In the sub-chronic oral toxicity study, it was demonstrated that treatment with WS400517 at doses up to and including 600 mg/kg bw/day for 90 days did not induce adverse effects to male and female rats.

This NOAEL does not necessitate any classification regarding repeated exposure according to European classification rules [REGULATION (EC) 1272/2008].