4-(4-isopropoxyphenylsulfonyl)phenol

Endpoint summary

Description of key information

D-8 was tested for repeated dose toxicity in a 90 day key study by oral gavage and a 28 day suporting study by administration in the diet. D-8 administered daily by oral gavage for 90 days to Wistar rats did not lead to any toxicologically adverse effects at dose levels of 5, 10 or 50 mg/kg bw/day. The no observed effect level (NOEL) was 50 mg/kg bw /day.

D-8 was tested for repeated dose toxicity in a 28-day study by administration of the test article in the diet to male and female F344 rats. Applied dose levels were 0, 120 ppm (low), 1.200 ppm (mid) and 12.000 ppm (high) for 4 weeks. Clinical observation (daily), body weight and food consumption measurements (weekly), hematology, urinalysis and blood chemistry measurements (Week 4 and 6), gross necropsy including organ weight measurement, and histopathological examination (Week 4 and 6) were performed. The no-effect-level (NOEL) was estimated as 120 ppm (10.9 mg/kg/day in males and 11.9 mg/kg/day in females).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-28 to 2009-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-09-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90;
- Age at study initiation: less than 9 weeks old;
- Weight at study initiation: The weight variation in animals involved in the study did not exceed 20 % of the mean weight for each sex (males 253-292 g, females 174-220 g)
- Fasting period before study: no
- Housing: Group caging (up to 5 animals/cage); Cage type: III; polypropylene/polycarbonate
- Diet: Animals will receive ssniff® SM R/M-Z+H “Autoclavable complete feed for rats and mice” produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum;
- Water: tap water from the municipal supply from 500 mL bottle, ad libitum;
- Acclimatisation: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C;
- Humidity: 30 - 70 % R.H.;
- Air changes: 15-20 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Intended dose levels are 5, 10 and 50 mg/kg bw/day.
The test item was formulated in the vehicle in concentrations of 1.25, 2.5 and 12.5 mg/mL.
The test item was administered by oral gavage on a 7 days/week basis. Control animals were treated concurrently with the vehicle only. A constant treatment volume of 4 mL/kg body weight was administered in all groups. The actual treatment volume was calculated according to the most recent body weight. Animals were be treated on the day of gross pathology.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC system: Merck-Hitachi LaChrom HPLC system
Analyses of dose formulations (concentration and homogeneity) were conducted on three occasions during the study (weeks 1, 6 and 13). All dose formulations were homogenous at all analytical occasions. No D-8 was detected in the vehicle control samples. In the test item samples, the measured concentrations ranged from 92 to 100% of nominal concentrations (1.25, 2.5, and 12.5 mg/mL). These results were considered suitable for the study purposes.

Duration of treatment / exposure:

90 Days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex and per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the available information from a 28-day feeding study with the test item. Based on growth depression in the high dose group of both sexes, increased liver weights accompanied by hepatic centrilobular swelling in the mid and high dose group and increased kindey weights accompanied by histopathological findings (i.a. calcium deposition in renal tubules in male animals) in the mid and high dose group the NOEL of this study was 10 mg/kg bw/day. Based on these results and considering that the test item is administered orally by gavage leading to higher peak concentrations and over 90 days the highest dose was established to be 50 mg/kg bw/day. Higher doses were expected to exceed the Maximum tolerated dose (MTD).
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, unusual respiratory pattern), occurrence of secretions and excretions, circulatory and central nervous system, somatomotor activity and behaviour pattern (clonic or tonic movements, stereotypies, bizarre behaviour), changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma

BODY WEIGHT: Yes
- Time schedule for examinations: For each animal, body weight was recorded on Day 0 (beginning of the experiment), then weekly during the treatment period with a precision of 1 g. On the day of gross pathology, fasted body weights were determined.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The food consumption was determined weekly by re-weighing the non consumed diet during the treatment period with a precision of 1 g.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The fundus of eyes of all animals was examined before treatment and in the control and high-dose groups (Groups 1 and 4) during week 13. As no treatment related alterations were found, no further, terminal evaluation was required.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Anaesthetic used for blood collection: Yes, pentobarbital
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table No.2 were examined.

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

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were assessed in all animals during week 13. General physical condition and behaviour of animals were tested. A modified Irwin test was performed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organs/tissues were removed and preserved in 10% buffered formalin solution for histological processing:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum and jejunum with Peyer’s patches, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, ovaries with oviduct, uterus with cervix, vagina, epididymes, brain (including cerebrum, cerebellum, pons and medulla oblongata), lachrymal gland with Harderian glands, seminal vesicle with coagulating glands, muscle (quadriceps), sciatic nerve and aorta.
The eyes with the optic nerves and the testes were preserved in modified Davidson fixative for histological processing.

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of all animals from the control and high dose groups. In the low and mid dose groups, the tissues with gross lesions were also processed histologically.

Statistics:

Statistical analysis was performed on the following data sets using SPSS PC+ software: body weight, food consumption, haematology, clinical chemistry, urinalysis and organ weight.
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was assessed by a Kolmogorov-Smirnov test.
In case of a not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied.
If there was a positive result, the inter-group comparisons were performed using a Mann-Whitney U-test.
Frequency of toxic response, pathological and histopathological findings and the mean daily food consumption by sex and dose were calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test-item related clinical signs observed following administration of D-8 by oral gavage, daily for 90 days, at dose levels up to and including 50 mg/kg bw/day.
One high dose male was noted to have limited use of limbs and decreased activity for 12 and 13/90 days, respectively (from day 65 to days 76 and 77, respectively). Based on the lack of clinical signs or adverse effects in other animals, this observation was considered incidental and not related to test item administration.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects were noted in the mean body weight and body weight gain values of the treated groups compared to control animals following daily administration of D-8 at dose levels of up to and including 50 mg/kg bw/day.
Slightly lower mean body weights with no statistical significance when compared to control animals were noted on occasion at 50 mg/kg bw/day in the male animals, and at all the dose levels tested in the female animals. The variations were minor (within -4%) and no significant differences were noted in the body weight gain values. In the absence of a consistent dose or genderrelated response, these changes were considered neither toxicologically significant, nor related to D-8 administration, but expected variations in the population of Wistar rats.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences in the mean daily food intake considered related to D-8 administration in any of the treated groups (5, 10, or 50 mg/kg bw/day, male or female) when compared to the control.
In the male animals, no statistically significant changes occurred in the animal feed intake. In the female animals, variations were observed on occasion in the daily mean food consumption, generally with slightly higher values in the treated groups at 10 or 50 mg/kg bw/day dose levels compared to controls. Although these variations have occasionally attained statistical significance (i.e. at weeks 6, 7, 9 and/or 11), the differences were minor and not considered toxicologically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects considered related to test item administration were observed during the ophthalmoscopy examination performed during week 13 on the control and high dose animals.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant differences between the control and test item-treated groups, or any adverse effects of D-8 on haematology parameters in the male and female animals.
Statistically significant variations were noted on occasion in the values of a few haematology parameters. At 10 and 50 mg/kg bw/day, the mean platelet thrombocyte volume (MPV) was higher than control in the male animals, but lower in the female animals, with no dose response. In addition, neutrophil value (NE%) was lower in the test-item treated males, with statistical significance at 5 and 10 mg/kg bw/day, but showed no significant differences in the female animals. Lymphocyte (LY%) value was slightly higher in the treated male animals, attaining statistical significance at 5 mg/kg bw/day, but not at the 10 and 50 mg/kg bw/day dose levels; LY% was comparable, or slightly lower in the treated female groups than control. Other parameters displayed minor variations without statistical significance or consistent dose or gender pattern in the treated groups when compared to control animals (i.e. monocyte MO%, basophil BA% or large unstained cells LUC%). As no consistent response was observed in correlation with the dose level or between male and female animals, the differences observed between the control and treated groups were considered to be incidental findings which were not related to treatment and with no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no relevant changes in the examined clinical chemistry parameters that could be attributed to D-8 administration.
Statistically significant variations of creatinine, concentrations of sodium (Na+), chloride (Cl-), calcium (Ca++), and/or phosphorus (Phos.), aspartate aminotransferase activity (AST) and albumine/globuline ratio (A/G) were noted in the treated animals compared to control. In the males, these variations were minor, showed no dose response, and consisted of higher mean creatinine values in the 10 and 50 mg/kg bw/day groups compared with control animals, higher Na+ at 5 mg/kg bw/day (but
lower at 50 mg/kg bw/day), higher Cl- at 5 and 50 mg/kg bw/day, higher Ca++ value in the low dose 5 mg/kg bw/day animals (but lower, with no statistical significance, at higher dose levels), and higher AST at 10 mg/kg bw/day, although lower, with no statistical significance, at 5 or 50 mg/kg bw/day. In the females, statistically significant, slight variations in comparison with the control group occurred at all the dose levels tested. At 5 mg/kg bw/day, higher Cl-, albumine and A/G were noted; in the mid dose group at 10 mg/kg bw/day, Cl-, Phos. and A/G mean values were higher than control, and lower Ca++ was observed; at 50 mg/kg bw/day, lower Na+, Cl- and Ca++ mean values were recorded. As no dose related response was observed and/or there was no consistent reaction in the male and female groups, these variations were neither considered toxicologically significant, nor indicated a test-item related etiology.

Urinalysis findings:

no effects observed

Description (incidence and severity):

D-8 administration administered by daily oral gavage for 90 days at up to and including 50 mg/kg bw/day did not result in any effects considered test-item related on the analyzed urinalysis parameters.
The pH was lower in the high dose 50 mg/kg bw/day males, the urinary volume higher in the mid dose 10 mg/kg bw/day males, and the specific gravity (SG) of urine lower in the mid dose 10 mg/kg bw/day female animals compared to controls, with no similar changes in the other gender. These variations were slight, showed no dose-dependent tendency, and there was inconsistency between sexes, therefore, the differences were not considered to be of toxicological importance in correlation with test item administration in the conditions of this study. The other minor variations observed did not attain statistical significance and/or were regarded as normal background changes.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant changes in the examined organ weight values (absolute and relative to the body and brain weights) noted after D-8 administration daily for 90 days, at up to and including 50 mg/kg bw/day.
No statistically significant differences were observed in the organ weights of the control and treated male animals. In the female animals, uterus and/or ovaries showed slightly higher absolute and relative to body and/or brain weights mean values at 50 mg/kg bw/day. However, as these changes were minor, did not show any dose response and were not correlated with pathological findings, they were considered incidental, not related to treatment and likely associated with physiological changes during the oestrus cycle of the females. All other examined organ weights (absolute and relative to the body and brain weights) were similar in the control and test item treated female animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no D-8-related macroscopic findings at a dose level up to 50 mg/kg/bw/day.
Dark red discoloration or pale area or pale mottling were noted in the lungs throughout all the groups, including controls, and occasionally dark red discoloration was observed in the thymus and seminal vesicle; these lesions were considered likely related to terminal procedures. Other macroscopic findings such as, but not limited to, dilatation of the renal pelvis, calculus on the mesentery, uterus in estrous cycle and/or ovarian cyst, small or enlarged testes and/or epididymides occurred on occasion and were regarded as incidental, not related to treatment in the conditions of this study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of D-8-related microscopic findings in the high dose 50 mg/kg bw/day animals or in the examined tissues and organs with macroscopic findings from the low and mid dose groups, at 5 and 10 mg/kg bw/day, respectively.
The microscopic findings recorded consisted of lesions such as, but not limited to, minimal diffuse extramedullary hematopoesis in the splenic red pulp, minimal, or mild vacuolation in the adrenals, minimal congestion and/or hemorrhage, minimal subacute inflammation, tubular cyst, tubular basophilia or pelvic dilatation in the kidneys, minimal dilatation of the glandular mucosa in stomach, or ovarian cyst. Tubular dilatation associated with reduced sperm content or tubular atrophy were observed in the testes, and ductal atrophy with reduced sperm content, or ductal cell debris were noted in the epididymides in two Low Dose males. Minimal mesenterial fibrolipoma (described macroscopically as yellow calculus on the mesentery) was recorded in one Low Dose female. These findings had either low incidence and/or severity, or were observed across control and dosed animals and thus were considered to be incidental and not related to D-8 administration.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose with no effects

Key result

Critical effects observed:

no

Conclusions:

In conclusion, D-8 administered daily by oral gavage for 90 days in Wistar rats did not lead to any toxicologically adverse effects at dose levels of 5, 10 or 50 mg/kg bw/day. Under the conditions of this study, the no observed effect level (NOEL) for D-8 is 50 mg/kg bw/day.

Executive summary:

A 90-day oral toxicity study according OECD TG 408 was performed with D-8 in male and female CRL:(WI) BR Wistar rats. A control and three dose groups (n= 10 animals per group and sex) were included in the study. The test item was administered daily by oral gavage, in concentrations of 0, 1.25, 2.5, and 12.5 mg/mL prepared in PEG 400 corresponding to 0, 5, 10 and 50 mg/kg bw/day doses levels, at a 4 mL/kg bw treatment volume. Stability and homogeneity of test item in this vehicle were analytically proven. Assessment of D-8 stability in PEG 400 vehicle at concentrations of approximately 1 to 250 mg/mL (LAB study code 08/775-316AN) indicated an up to 24-hour stability at room temperature, and an up to 72-hour stability when stored refrigerated at 5-3 °C, with a recovery within the acceptable range of 100 ± 10% (96-102 %, and 99-104 %, respectively). Analyses of dose formulations (concentration and homogeneity) were conducted on three occasions during the study (weeks 1, 6 and 13). All dose formulations were homogenous at all analytical occasions. No D-8 was detected in the vehicle control samples. In the test item samples, the measured concentrations ranged from 92 to 100% of nominal concentrations (1.25, 2.5, and 12.5 mg/mL). These results were considered suitable for the study purposes. Animals were inspected for signs of morbidity and mortality twice daily (. Clinical observations for signs of ill health or reaction to treatment were made once daily. A detailed clinical examination was made before the first treatment, then once weekly during the study with the examinations performed with animals removed from the cage. During week 13, animal sensory reactivity to different type of stimuli, grip strength, motor activity, general physical condition and animal behaviour were tested and a modified Irwin test was performed in order to evaluate any possible neurotoxicity effects. Ophthalmoscopy examination of the control and high dose animals was conducted during week 13. Body weight and food consumption were measured weekly. Clinical pathology examinations (haematology with evaluation of the blood clotting times, clinical chemistry and urinalysis) and gross necropsy were conducted at the end of the treatment period. The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, uterus including cervix, ovaries and thymus were determined. A full histopathological examination was performed on the selected list of preserved organs and tissues of the animals of Groups 1 (Control) and 4 (High dose), and on abnormal tissues from Low and Mid dose groups.

Results and Discussions

D-8 caused no mortality at up to and including 50 mg/kg bw/day in CRL:(WI)BR rats. There were no test-item related clinical signs following administration of D-8 by oral gavage, daily for 90 days. The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative for neurotoxicity were observed. No effects were noted during the ophthalmoscopy evaluation performed on completion of treatment. There were no toxicologically significant changes in body weight, body weight gain or animal feed intake between the control and test item treated groups. Differences were noted in the test item treated groups compared to controls in a few clinical pathology parameters evaluated prior to necropsy (haematology, coagulation, clinical chemistry and/or urinalysis). Although occasionally these changes were statistically significant, no dose related response was observed, the differences were minor and/or there was no consistent reaction in the male and female groups. These changes were not considered toxicologically significant, nor indicated a test item related etiology. There were no macroscopic or microscopic adverse findings, no statistically or toxicologically significant changes in organ weight values or any pathology changes that could be ascribed to test item administration. In conclusion, D-8 administered daily by oral gavage for 90 days in Wistar rats did not lead to any toxicologically adverse effects at dose levels of 5, 10 or 50 mg/kg bw/day. Under the conditions of this study, the no observed effect level (NOEL) for D-8 is 50 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD TG 408

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The performance of a repeated dose test for toxicity by inhalation route is scientifically not justified. REACh Regulation (EC) No 1907/2006, Annex IX, Sect. 8.6.2, Col. 2, states as follows: “8.6.2: Testing by inhalation route is appropriate if exposure on humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of inhalable size.” During use D-8 is included in three-dimensional matrices of resinous material and completely retained. Therefore, exposure to aerosols, particles or droplets of inhalable size is unlikely and inhalation practically negligible. For more details on exposure assessment see the risk assessment report in section 13. Further, physico-chemical properties of D-8, including particle size determination, suggest no evidence of a significant absorption by inhalation. The substance is practically non-volatile, with vapour pressure determined below 1.0E-05 Pa at 27 °C and the mean volumetric diameter of particles was determined to be 5.12 µm. Therefore, availability of D-8 in air and the formation of inhalable particles are not very likely. The acute inhalation toxicity testing showed no local or systemic toxicity. Neither skin nor dermal irritation tests showed signs of systemic effects by absorption through skin or mucous membranes. A 90 day oral repeated dose toxicity study in rats revealed a NOEL of 50 mg/kg bw/day. In summary, based on exposure assessment and available toxicity tests, further repeated dose testing of D-8 by inhalation is scientifically not justified.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The performance of a repeated dose test for toxicity by inhalation route is scientifically not justified. REACh Regulation (EC) No 1907/2006, Annex IX, Sect. 8.6.2, Col. 2, states as follows: “8.6.2: Testing by inhalation route is appropriate if exposure on humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of inhalable size.” During use D-8 is included in three-dimensional matrices of resinous material and completely retained. Therefore, exposure to aerosols, particles or droplets of inhalable size is unlikely and inhalation practically negligible. For more details on exposure assessment see the risk assessment report in section 13. Further, physico-chemical properties of D-8, including particle size determination, suggest no evidence of a significant absorption by inhalation. The substance is practically non-volatile, with vapour pressure determined below 1.0E-05 Pa at 27 °C and the mean volumetric diameter of particles was determined to be 5.12 µm. Therefore, availability of D-8 in air and the formation of inhalable particles are not very likely. The acute inhalation toxicity testing showed no local or systemic toxicity. Neither skin nor dermal irritation tests showed signs of systemic effects by absorption through skin or mucous membranes. A 90 day oral repeated dose toxicity study in rats revealed a NOEL of 50 mg/kg bw/day. In summary, based on exposure assessment and available toxicity tests, further repeated dose testing of D-8 by inhalation is scientifically not justified.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Performance of a repeated dose test for toxicity by the dermal route is scientifically not justified. REACh Regulation (EC) No 1907/2006, Annex IX, Sect. 8.6.2, Col. 2, states as follows: “8.6.2: Testing by the dermal route is appropriate if: - (1) skin contact in production and/or use is likely; and - (2) the physicochemical properties suggest a significant rate of absorption through the skin; and - (3) one of the following conditions is met: - toxicity is observed in the acute dermal toxicity test at lower doses than in the oral toxicity test, or - systemic effects or other evidence of absorption is observed in skin and/or eye irritation studies, or - significant dermal toxicity or dermal penetration is recognised for structurally-related substances.” Use of formulated D-8 is unlikely to result in exposure via the dermal route. During use D-8 is included in three-dimensional matrices of resinous material and completely retained. Therefore, exposure via the dermal route is practically negligible. For more details on exposure assessment see the risk assessment report in section 13. Further, physico-chemical properties of D-8 suggest no evidence of a significant absorption by the dermal route. The acute dermal toxicity testing showed no local or systemic toxicity. Neither skin nor dermal irritation tests showed signs of systemic effects by absorption through skin or mucous membranes. A 90 day oral repeated dose toxicity study in rats showed a NOEL of 50 mg/kg bw/day. Acute oral administration at dose levels up to 5000 mg/kg bw/day (limit dose) did not reveal any toxicological effects. In summary, based on exposure assessment and available toxicity tests, further repeated dose testing of D-8 by the dermal route is scientifically not justified.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Performance of a repeated dose test for toxicity by the dermal route is scientifically not justified. REACh Regulation (EC) No 1907/2006, Annex IX, Sect. 8.6.2, Col. 2, states as follows: “8.6.2: Testing by the dermal route is appropriate if: - (1) skin contact in production and/or use is likely; and - (2) the physicochemical properties suggest a significant rate of absorption through the skin; and - (3) one of the following conditions is met: - toxicity is observed in the acute dermal toxicity test at lower doses than in the oral toxicity test, or - systemic effects or other evidence of absorption is observed in skin and/or eye irritation studies, or - significant dermal toxicity or dermal penetration is recognised for structurally-related substances.” Use of formulated D-8 is unlikely to result in exposure via the dermal route. During use D-8 is included in three-dimensional matrices of resinous material and completely retained. Therefore, exposure via the dermal route is practically negligible. For more details on exposure assessment see the risk assessment report in section 13. Further, physico-chemical properties of D-8 suggest no evidence of a significant absorption by the dermal route. The acute dermal toxicity testing showed no local or systemic toxicity. Neither skin nor dermal irritation tests showed signs of systemic effects by absorption through skin or mucous membranes. A 90 day oral repeated dose toxicity study in rats showed a NOEL of 50 mg/kg bw/day. Acute oral administration at dose levels up to 5000 mg/kg bw/day (limit dose) did not reveal any toxicological effects. In summary, based on exposure assessment and available toxicity tests, further repeated dose testing of D-8 by the dermal route is scientifically not justified.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 90-day oral toxicity study according OECD TG 408 was performed with D-8 in male and female CRL:(WI) BR Wistar rats. A control and three dose groups (n= 10 animals per group and sex) were included in the study. The test item was administered daily by oral gavage, in concentrations of 0, 1.25, 2.5, and 12.5 mg/mL prepared in PEG 400 corresponding to 0, 5, 10 and 50 mg/kg bw/day doses levels, at a 4 mL/kg bw treatment volume. Stability and homogeneity of test item in this vehicle were analytically proven. Assessment of D-8 stability in PEG 400 vehicle at concentrations of approximately 1 to 250 mg/mL (LAB study code 08/775-316AN) indicated an up to 24-hour stability at room temperature, and an up to 72-hour stability when stored refrigerated at 5-3 °C, with a recovery within the acceptable range of 100 ± 10% (96-102 %, and 99-104 %, respectively). Analyses of dose formulations (concentration and homogeneity) were conducted on three occasions during the study (weeks 1, 6 and 13). All dose formulations were homogenous at all analytical occasions. No D-8 was detected in the vehicle control samples. In the test item samples, the measured concentrations ranged from 92 to 100% of nominal concentrations (1.25, 2.5, and 12.5 mg/mL). These results were considered suitable for the study purposes. Animals were inspected for signs of morbidity and mortality twice daily (. Clinical observations for signs of ill health or reaction to treatment were made once daily. A detailed clinical examination was made before the first treatment, then once weekly during the study with the examinations performed with animals removed from the cage. During week 13, animal sensory reactivity to different type of stimuli, grip strength, motor activity, general physical condition and animal behaviour were tested and a modified Irwin test was performed in order to evaluate any possible neurotoxicity effects. Ophthalmoscopy examination of the control and high dose animals was conducted during week 13. Body weight and food consumption were measured weekly. Clinical pathology examinations (haematology with evaluation of the blood clotting times, clinical chemistry and urinalysis) and gross necropsy were conducted at the end of the treatment period. The absolute and relative organ weights of adrenals, brain, epididymides, heart, liver, kidney, spleen, testes, uterus including cervix, ovaries and thymus were determined. A full histopathological examination was performed on the selected list of preserved organs and tissues of the animals of Groups 1 (Control) and 4 (High dose), and on abnormal tissues from Low and Mid dose groups.

Results and Discussions

D-8 caused no mortality at up to and including 50 mg/kg bw/day in CRL:(WI)BR rats. There were no test-item related clinical signs following administration of D-8 by oral gavage, daily for 90 days. The behaviour and the general condition of the test animals were normal during the study. There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative for neurotoxicity were observed. No effects were noted during the ophthalmoscopy evaluation performed on completion of treatment. There were no toxicologically significant changes in body weight, body weight gain or animal feed intake between the control and test item treated groups. Differences were noted in the test item treated groups compared to controls in a few clinical pathology parameters evaluated prior to necropsy (haematology, coagulation, clinical chemistry and/or urinalysis). Although occasionally these changes were statistically significant, no dose related response was observed, the differences were minor and/or there was no consistent reaction in the male and female groups. These changes were not considered toxicologically significant, nor indicated a test item related etiology. There were no macroscopic or microscopic adverse findings, no statistically or toxicologically significant changes in organ weight values or any pathology changes that could be ascribed to test item administration. In conclusion, D-8 administered daily by oral gavage for 90 days in Wistar rats did not lead to any toxicologically adverse effects at dose levels of 5, 10 or 50 mg/kg bw/day. Under the conditions of this study, the no observed effect level (NOEL) for D-8 is 50 mg/kg bw/day.

D-8 was tested for repeated dose toxicity in a 28-day study according to OECD TG 407 (Takaori, 1988). The test article was administrated in the diet to male and female F344 rats. Applied dose levels were 0, 120 ppm (low), 1.200 ppm (mid) and 12.000 ppm (high) (corresponding to approximately 10, 100 and 1000 mg/kg bw/d) for 4 weeks. Clinical observation (daily), body weight and food consumption measurements (weekly), hematology, urinalysis and blood chemistry measurements (Week 4 and 6), gross necropsy including organ weight measurement, and histopathological examination (Week 4 and 6) were performed. A significant decrease of body weight, body weight gain and feed intake between the control and the high dose animals was noted. Investigation of organ weights revealed a statistically significant decrease of adrenals weight (abs.) in males of the high dose group. In addition, a statistically significant decrease of ovary weight (abs. & rel.) was observed at 1000 mg/kg bw/d. The absolute testis weights were also decreased but the testis weight relative to organ weight was increased in animals of the high dose group. After two weeks of recovery the decreased testis weight (abs.) was still present whereas the testis weight ratios recovered due to the regeneration of body weights in animals at 1000 mg/kg bw/day. Additionally, no differences in ovary weights between control and high dose females were observed. Liver weight and its ratio to body weight were increased in the mid and/or high dose groups. Kidney weight ratio was also increased in both sexes of the high dose group. Histopathological examinations revealed hepatic centrilobular swelling in both sexes of the high dose group. Calcium deposition in renal tubules, which is normal finding in this strain of female rats, was also found in males of the mid and high dose groups. Eosinophilic body, which usually present in proximal tubules of kidney in male rats of this strain, was not observed in males of the high dose group. The incidence of tubular regeneration was higher in males of the high dose group than that of the control group. Since all findings in organs putatively affected by the hormonal system occurred at excessive systemic toxicity in the high dose groups only and are mostly reversible after two weeks of recovery they are considered of no toxicological relevance. Based on these data, it was concluded that the target organs of D-8 are liver, kidney, cecum, spleen and bone marrow and the no adverse effect level (NOAEL) was 120 ppm (10.9 mg/kg bw/day in females.)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.