Diisodecyl sebacate

Endpoint summary

Description of key information

A guideline OECD 408 90-day repeated-dose toxicity study was performed using DIDS. No effects were observed in female rats at the highest dose (1000 mg/kg bw/day). In the males, due to the observed histopathological changes in the kidney, which most probably corresponds to Alpha 2u globulin deposition (an adverse nephropathy for male rats only) and is not indicative of a risk for human health.Whilst the rat-specific NOAEL for CEREPLAS DIDS in the males is considered to be 300 mg/kg bw/day, the NOAELfor all the human-relevant endpoints is considered to be 1000 mg/kg bw/day, basedon the results of this study.
No adverse effects were seen in a 14-day oral study in rats administered DIDA, presumably by gavage, at 1000 mg/kg bw/day (Conning, 1970). In a chronic dietary study, no adverse effects were seen when rats were given DOS at about 10 mg/kg bw/day for up to 19 months (Le Breton, 1952-7).
The secondary literature describes a subchronic inhalation study on DEHS, in which groups of 12 rats were exposed to up to 250 mg/m3 for 13 wk. Although the final analysis of the data was not formalised, no significant effects appeared evident [no further details given] (Swift, 1983). 
No repeated dose dermal studies were available on DIDS (or DIDA or DEHS/DOS).

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/04/2019 - 22/04/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See Principles of Methods

Principles of method if other than guideline:

Deviations

Due to a miscalculation during the preparations of the formulas, for one day (30 May 2019, Day 22), Top dose animals received only 60% of the nominal concentration. Given that for the rest of the 28-days long treatment period, the animals received the correct dosage, this deviation is considered to have no impact on the outcome of the study and interpretation of the results.

Due to technical reasons, the actual relative humidity range was 29– 60% instead of 3070 % as it was indicated in the Study Plan. This deviation is considered to have no impact on the outcome of the study and interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Standard strain for repeated-dose studies

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals approximately 10 weeks old at time of treatment. Acclimation period was 13 days.

Route of administration:

oral: gavage

Details on route of administration:

The dosing formulations were administered daily starting from Day 0 for 28 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw was administered to all animals in all groups. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Vehicle:

propylene glycol

Details on oral exposure:

The dosing formulations were administered daily starting from Day 0 for 28 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw was administered to all animals in all groups. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Control animals were treated concurrently with the vehicle only (1% Tween 80 in propylene glycol). Animals were not treated on the day of necropsy.

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed in the Analytical Laboratory of Charles River Laboratories Hungary Kft. using a validated HPLC-UV method (Charles River Laboratories Hungary Kft. study code 18/296-316AN [5]).

Top, middle and bottom duplicate samples were taken from test item formulations once during the study, one set to analyse and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken in duplicate from the middle of the vehicle control solution for concentration measurement.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

General clinical observations were made at least twice daily, at the beginning and towards the end of the working day as practical, with the exception of Day 28, when only a detailed observation was performed. From Day 16, some non-adverse clinical changes had been seen in the animals, hence an additional post-dose clinical observation was added to the daily routine.

Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

The animals were monitored for any clinical signs, including pertinent behavioural changes, signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep or coma.

Body weight measurement

Body weight was recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 27 (last treatment day) and prior to necropsy (fasted) on Day 28.

Food consumption measurement

The determination of food consumption was performed for all groups once a week. The remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Weekly food consumption was calculated.

Sacrifice and pathology:

On Day 28, blood samples for clinical pathology evaluation (haematology and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia.

After an overnight period of food deprivation of animals, two blood samples were collected; one for haematology (in tubes with K3-EDTA, 1.6 mg/mL blood) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Terminal procedures and Macroscopic evaluation

Gross necropsy was performed on all animals at the end of treatment period on Day 28. The animals were euthanized by exsanguination under pentobarbital anaesthesia.

After exsanguination, the external appearance was examined, the cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

On completion of the macroscopic observation and organ weight measurements, the carcasses were discarded. No tissue preservation was performed.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant body weight changes in the 1000 mg/kg bw/day dose group (males/females) when compared to the control rats at any occasion.

Lower body weight gains were observed from Day 7 up to Day 14, and from Day 21 up to Day 27 in the males of the 1000 mg/kg bw/day dose group, reaching statistical significance in the Day 7- Day 14 period (p < 0.01). The overall body gain difference between the control and males receiving 1000 mg/kg bw/day were slightly less than 10 percent (-9.8%) throughout the study. Significantly higher body weight changes were observed in the females receiving 1000 mg/kg bw/day between Day 21 and Day 27 when compared to the control.

These findings correlate well with the similar food consumption findings (decreased food consumption at the onset of treatment in males receiving 1000 mg/kg bw/day, increased food consumption in the last week of treatment in females receiving 1000 mg/kg bw/day).

However, the growth curve for both sexes correlates well with the control group and historical control data, therefore these changes, while the correlation with the treatment could not be excluded, are not considered to be an adverse effect.

In summary, there were no adverse effects on body weight or on body weight gain that were ascribed to the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first week, lower food consumption was measured by 8-8.6% in the males and females receiving 1000 mg/kg bw/day. The animals showed adaptation to the test item, but in the males, a much smaller (-3.6%) difference was still observed during the second week. These changes were considered to be test item related.

Higher food consumption (20.7%) was observed in the females receiving 1000 mg/kg bw/day; this change was considered to be incidental, and most probably due to the low number of animals in the study.

Haematological findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment noted in the haematology parameters.

Minor variations were observed, predominantly in the exposed females; HGB, MCHC, relative basophil cells, RDW, and LUC values, attaining statistical significance. The changes were considered to be incidental and related to individual variation. All data is within the normal range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment noted in the clinical chemistry parameters.

Statistically significant values were recorded only for ALT/GPT values of the females receiving 1000 mg/kg bw/day. When compared to the control group animals, the difference was only 40%, which is consistent with the organ weight and histopathological changes of the livers, but not indicating any adversity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed on the liver and kidney weight for both sexes.

The absolute and relative liver weights in the 1000 mg/kg bw/day dose group were significantly (p<0.01) increased in both sexes, by about 17% in males and by about 54% in females.

Kidney weights were increased in the 1000 mg/kg bw/day dose group in both sexes by about 13%, reaching statistical significance in the males only (p<0.05).

Furthermore, adrenal glands were significantly higher in the females of the 1000 mg/kg bw/day dose group.

Based on the relative change in the organ weights and the histopathological results, the changes in the liver were considered to be adaptive responses to the treatment. Without histopathological examination, the adversity of the kidney weight changes could not be determined.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed.

Bilateral small size in the adrenal in 1/5 male animals and small right adrenal in 1/5 male animals was recorded for the 1000 mg/kg bw/day dose group The toxicological significance of these findings is unclear in the absence of histopathological examination, and in the absence of similar changes in the females receiving 1000 mg/kg bw/day.

All other observed changes were considered incidental or within the normal historical background.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the histopathological examination of the livers, minimal to mild centrilobular hypertrophy was observed in the males and females of the 1000 mg/kg bw/day dose group, indicating an adaptive change most probably due to the increased xenobiotic metabolism activity of the liver. The kidneys were not examined.

Key result

Dose descriptor:

dose level:

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

not measured/tested

Key result

Critical effects observed:

no

Conclusions:

Following administration of a 1000 mg/kg bw/day of the test material, effects were observed that were attributed to the test item. These included:

- Lower food consumption in male animals
- Increase in liver and kidney weights
- Increase in ALT/GPT associated with the increase in organ weights

Executive summary:

CEREPLAS DIDS administered by oral gavage to Wistar rats for 28 consecutive days at the dose level of 1000 mg/kg body weight/day in 1% Tween 80 in propylene glycol at a dose volume of 5 mL/kg body weight, caused the following effects:

Increase of the liver and kidney weights were recorded in the animals receiving 1000 mg/kg bw/day, with no correlating adverse histopathology findings. The liver weight increase in females, which was about 54%, might limit the practicability of

testing at higher dose levels; the liver weight increase in the males was approximately 16%, which is expected to be compatible with longer term administration.

In selecting dose levels for the following OECD 408 study, the highest dose is proposed to be 1000 mg/kg bw/day; lower dose levels should be set to ensure that a NOAEL can be determined.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-07-2019 to 28-10-2019

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)

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
Hygienic level: SPF at arrival, standard laboratory conditions during the study.
Number of animals: 40 male and 40 female rats, 10 animals/group/sex,
4 groups. Additional 8 spare animal/sex were ordered.
Age of animals: Young adult rats, approx. 6-7 weeks old at start of treatment.
Body weight: Males: 203 – 237 g, females: 170 – 216 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Animal receipt: 18 July 2019
Acclimation period: 5 days (males) 12 days (females)
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Cage type: Type II and/or III polycarbonate
Bedding and nesting: SAFE ¾ -S (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany) Bedding for Laboratory Animals and nest building material (SAFE crinklets natural (produced by J. Rettenmaier & Söhne GmbH + Co.KG, Germany)) were available to animals during the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.8 – 24.7 °C
Relative humidity: 30 – 67 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were housed 2 or 3 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.

Route of administration:

oral: gavage

Vehicle:

other: 1% Tween 80 in propylene glycol

Details on oral exposure:

The dose formulations were administered daily starting from Day 0 for 91 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw/day was administered to all animals. The actual volumes to be administered were calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation) in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared up to 4 days before use (formulation were kept closed, at room temperature until use when stored). Stability of the test item in the vehicle was assessed in the conditions employed on the study during the analytical method validation [4]. In that study, the formulation samples in the 10-250 mg/mL concentration range (using 1% Tween 80 in propylene glycol as vehicle) were proven as being stable for at least 4 days when stored at room temperature.

Analysis of test item formulations for concentration and homogeneity was performed using an HPLC-UV method in the Analytical Laboratory of Charles River Laboratories Hungary Kft. Top, middle and bottom duplicate samples were taken from test item formulations four times during the study (during first, fifth, ninth and last week of the treatment), one set to analyse and one set as a back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 15 % of the nominal concentration.

Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) to be less than 10%.

Any sample not required for analysis were discarded following acceptance of the results of the formulation analysis by the Contributing Scientist and Study Director.

Duration of treatment / exposure:

90 days

Frequency of treatment:

A constant dose volume of 5 mL/kg bw/day was administered to all animals

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes

Details on study design:

The dose levels were set by the Sponsor in consultation with the Study Director, based on the available data and information from previous experimental work, including the results of a 28-Day Oral (gavage) Dose Range Finding Toxicity Study in Wistar Rats (Citoxlab study code: 18/296-101PE) [7].
In that study, no adverse effect was observed in the test item treated group (1000 mg/kg bw/days). Increase of the liver and kidney weights in the test item treated group were recorded, with no correlating adverse liver histopathology findings.
Based on the results from the above study, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study. The aim was to use a maximum of 1000 mg/kg bw/day to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
The oral route was selected as it is one of the possible routes of human exposure.

The dose formulations were administered daily starting from Day 0 for 91 consecutive days by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume of 5 mL/kg bw/day was administered to all animals. The actual volumes to be administered were calculated and adjusted based on the most recent individual body weight. Control animals were treated concurrently with the vehicle only.

Observations and examinations performed and frequency:

Mortality and clinical observations
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made at least once a day at approximately the same time with minor variations as practical.
Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on Day 0 male/female) and weekly thereafter, in the morning hours (am) and once before necropsy.
Observations were performed on the skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (ex. hunchback posture, etc.), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.


Ophthalmology evaluation
Ophthalmoscopic examination was conducted in all animals before treatment (Day -1 or -2), and in the Control group and High dose group animals, during Week 12 (Day 86). Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL cyclopentolate hydrochloride) into the conjunctival sac. The evaluation was performed using an Omega 500 ophthalmoscope. As in Week 12 no treatment related alterations were found in the Control group and High dose group animals, the remaining animals were not examined at termination.


Body weight measurements
Body weights were recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 90, and prior to necropsy, fasted on Day 91.


Food consumption measurements
The determination of food consumption was performed for all groups once a week.

The food was measured on Day 0 then the remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Daily food consumption was calculated for reporting purposes.


Neurological assessment (Functional Observation Battery and SMART)
Towards the end of the treatment period, during Week 11/12, all animals were examined in the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment.

Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.

A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioural and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as, body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.

To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were painted for any possible additional measurements.

Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.

Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.


Examination of vaginal smears

Sacrifice and pathology:

At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all animals. Food was withdrawn during the overnight urine collection

Other examinations:

After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with
K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry

Statistics:

Data were collected using the software PROVANTIS v.9. Group means and standard deviations were calculated from numerical data obtained in the study.

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 software package (within the validated Provantis system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.

The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Alopecia (on the neck or on the hindlimbs) was observed in 2 out of 10 males receiving 1000 mg/kg/day. These changes were most probably incidental, and not considered to be related to treatment.
In one female receiving 1000 mg/kg/day, tonic convulsion was observed on Day 79, 80, 82, and 88. As no other clinical signs were associated with this symptom, and as no abnormality was found during the necropsy, these findings were considered to be incidental, and while they might be related to the treatment, they are not regarded to be related to treatment

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on body weight or body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The measured food consumption values in the test item- treated groups were comparable to the control groups.
The observed statistical differences in certain periods were considered to be random differences, unrelated to the treatment, and were not considered to be an adverse effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the haematology parameters. The observed statistically significant differences were considered to be incidental, were not related to dose and/or all recorded values were near or within the historical control ranges. These differences were considered not to reflect an adverse effect of the test item. These incidental statistical differences can be seen in Table 6 and 7.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few clinical chemistry parameters were significantly different in the males and females in the mid and high dose groups. Generally, there was no dose response or consistency in the parameters between the sexes. The differences relative to the historical control range indicate that these are not adverse changes.
There were no statistical differences in clinical chemistry parameters in both males and females in the low dose groups compared to the Controls.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher urine volume, outside of the historical control range, was measured in the males receiving 1000 mg/kg/day. It is considered to be a treatment-related effect, in connection with the observed macroscopic and histopathological observations in the kidneys. Consequently, the higher volume of urine resulted in lower specific gravity and fewer observed urine crystals in this group.
The remaining urinalysis parameters were comparable to the concurrent or historical controls in all treated groups. The urinary pH in females receiving 1000 mg/kg/day showed statistical increases compared to controls, but the control values were higher than other recent 90-day rat studies; the values in males and females receiving 1000 mg/kg/day are considered to be normal. Other variations occurred without a dose response, were unrelated to treatment and considered to be normal.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At the functional observation battery (FOB) performed at the end of exposure (Week 11/12), there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or treated groups.
No treatment-related effects were observed in locomotor activity measured at the end of the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in kidney and liver weights in both males and females receiving either 300 or 1000 mg/kg/day.
The kidney weights (both absolute and relative to body and brain) were statistically significantly higher than Controls in both males and females receiving either 300 or 1000 mg/kg/day.
The liver weights (both absolute and relative to body and brain) were statistically significantly higher than Controls in males receiving either 300 or 1000 mg/kg/dayand in females receiving 1000 mg/kg/day.
The thyroid/parathyroid weight (absolute and relative to body and brain) were statistically significantly higher than Controls in males receiving 300 mg/kg/day, but histopathology did not reveal any microscopic findings attributed to the administration of the test item.
No organ weight change was seen in males of females receiving 100 mg/kg/day.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic findings were detected in the kidneys of males receiving 1000 mg/kg/day and in the liver of females receiving 1000 mg/kg/day.

Kidney
In the kidney of males receiving 1000 mg/kg/day, tubular basophilia (4/10), tubular dilatation (5/10) and presence of hyaline droplets in the tubules (4/10) were present and were correlated with organ weight changes. The spectrum of changes observed in the kidney are likely to be due to Alpha 2u globulin nephropathy.. The positive identification of hyaline droplets by special staining or immunohistochemistry for Alpha 2u globulin (Frazier et al 2012) would be required to confirm this conclusion. Similar changes were not observed in females receiving 1000 mg/kg/day.

Liver
In the liver of females receiving either 1000 mg/kg/day, minimal hepatocellular hypertrophy (8/10) and minimal hepatocellular vacuolation (8/10) were present that were correlated with organ weight changes. These findings were considered to be treatment related, non-adverse, adaptive changes.
The other findings (focal necrosis and haemorrhage in the adrenals and in the liver, luminal dilation in the uterus, tubular dilatation in the testis, reduced sperm content and intraluminal debris in the epididymis, hyaline casts and pelvic dilatation in the kidneys, harderian metaplasia in the lacrimal glands, ectopic tissue in the liver, cell infiltrate in the interstitium of the lungs and prostate, congestion/haemorrhage in the thymus and in the vaginal mucosa) were considered as incidental or to be within the background range.
In summary, for the organ weight and histology data, the liver weight changes in both males and females receiving either 300 or 1000 mg/kg/day were attributed to an adaptive response, based on the lack of histological changes in the males and non-adverse hepatocellular hypertrophy seen in females. Kidney weights in females receiving 1000 mg/kg/day were also increased with no histological changes, and were ascribed to an adaptive change. Kidneys in males receiving 1000 mg/kg/day did show histological changes consistent with Alpha 2u globulin nephropathy, which is adverse for male rats only; in the context of a human safety study, Alpha 2u globulin nephropathy in male rats is not indicative of a risk for human health.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

Critical effects observed:

no

Neurological assessment (functional observation battery)

Table 1:Grip strength means (males)

Grip strength (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Forelimbs (g)	1874.1	1948.9	1942.5	1816.9
	Differences from control (%)	4	3.7	-3.1
	Historical control data	1397 – 2438
Hind limbs (g)	683.3	754.7	790.2	692
	Differences from control (%)	10.4	15.6	1.3
	Historical control data	286 – 989

 

Table 2:Grip strength means (females)

Grip strength (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Forelimbs (g)	1517.9	1494.3	1573.5	1449.7
	Differences from control (%)	-1.6	3.7	-4.5
	Historical control data	1043 – 1765
Hind limbs (g)	614	687.4	651.4	691.9
	Differences from control (%)	12	6.1	12.7
	Historical control data	189 – 824

 

Table 3:Splay test means (males)

Splay test (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Distance between hindpaws (mm)	72.4	78.8	78.7	82.3
	Differences from control (%)	8.8	8.8	13.7
	Historical control data	33 – 133

 

Table 4:Splay test means (females)

Splay test (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Distance between hindpaws (mm)	65.3	71.3	58.6	65.3
	Differences from control (%)	9.1	-10.2	0
	Historical control data	27   – 138

 

 

Haematology

Table 5:Selected haematology parameters in males

 

Haematology (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Red blood count (M/µL)	8.750	8.695	8.56		8.362*
	Differences from control (%)	-0.6	-2.2		-4.4
	Historical control data	8.07 – 9.97
Haematocrit (%)	45.27	44.39		43.76^+		43.68^+
	Differences from control (%)	-1.9		-3.3		-3.5
	Historical control data	40.0 – 49.5
Mean Corpuscular Volume (fL)	51.79	51.08		51.15		52.26
	Differences from control (%)	-1.4		-1.2		0.9
	Historical control data	47.3 – 55.1
Red Cell volume (%)	12.66	13.19		13.36*		13.17
	Differences from control (%)	4.2		5.5		4
	Historical control data	11.6 – 14.4
Mean Platelet Thrombocyte volume (fL)	7.19	7.37		7.75		8.2*
	Differences from control (%)	2.5		7.8		14
	Historical control data	6.1 – 15.3
Reticulocytes (%)	2.01	2.06		2.21		2.41^++
	Differences from control (%)	2.5		10		19.9
	Historical control data	1.4 – 3.2
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two sided test. Values with significant differences are indicated with bold font.

 

 

Table 6:Selected haematology parameters in females

 

Haematology (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Red blood count (M/µL)	8.205	8.138	8.197		8.121
	Differences from control (%)	-0.8	-0.1		-1
	Historical control data	7.41 – 9.16
Haematocrit (%)	44.65	43.89		44.91		42.38
	Differences from control (%)	-1.7		0.6		-5.1
	Historical control data	41.1 – 48.5
Mean Corpuscular Volume (fL)	54.41	53.98		54.89		52.2*
	Differences from control (%)	-0.8		0.9		-4.1
	Historical control data	48.9 - -57.0
Red Cell volume (%)	12.63	12.07		12.12		12.6
	Differences from control (%)	-4.4		-4		-0.2
	Historical control data	10.4 – 14.1
Mean Platelet Thrombocyte volume (fL)	8.64	7.51		8.03		8.64
	Differences from control (%)	-13		-7		0
	Historical control data	6.0 – 9.4
Reticulocytes (%)	2.5	2.44		2.38		2.4
	Differences from control (%)	-2.2		-4.9		-4
	Historical control data	1.4 – 5.8
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Summary tables are given in Appendix 1.4.1, individual data in Appendix 2.4.1.

 

Coagulation parameters

Table 7:Selected coagulation parameters in males

 

Coagulation (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
PTProthrombin Time (sec)	9.63	9.6		9.69		9.49**
	Differences from control (%)	-0.3		0.6		-1.5
	Historical control data	9.2 – 10.5
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Table 8:Selected coagulation parameters in females

 

Coagulation (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
PTProthrombin Time (sec)	9.57	9.36		9.41		9.04
	Differences from control (%)	-2.2		-1.6		-5.5
	Historical control data	8.8 – 9.9
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Summary tables are given in Appendix 1.4.1., individual data in Appendix 2.4.1.

Clinical chemistry

Table 9:Selected clinical chemistry parameters in males

 

Clinical Chemistry (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Glucose (mmol/L)	8.91	8.44	8.78		8.19
	Differences from control (%)	-5.3	-1.5		-8.1
	Historical control data	7.05 – 12.68
Cholesterol (mmol/L)	1.736	1.799		1.682		1.769
	Differences from control (%)	3.6		-3.1		1.9
	Historical control data	1.16 – 2.73
Phosphorus (mmol/L)	2.49	2.64		2.705		2.853*
	Differences from control (%)	6		8.6		14.6
	Historical control data	1.93 – 3.02
Sodium (mmol/L)	140.8	141.8		142.5*		143.3*
	Differences from control (%)	0.7		1.2		1.7
	Historical control data	140.5 – 151.9
Bile acids (umol/L)	15.002	17.311		18.613		17.411
	Differences from control (%)	15.4		24.1		16.1
	Historical control data	5.53 – 35.56
HDL (mmol/L)	1.073	1.071		1.016		1.084
	Differences from control (%)	-0.2		-5.3		1
	Historical control data	NA
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

 

 

Table 10:Selected clinical chemistry parameter in females

 

Clinical Chemistry (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Glucose (mmol/L)	9.37	8.6	8		6.83**
	Differences from control (%)	-8.2	-14.6		-27.1
	Historical control data	4.96 – 12.78
Cholesterol (mmol/L)	1.485	1.496		1.551		2.050**
	Differences from control (%)	0.7		4.4		38
	Historical control data	1.16 – 3.00
Phosphorus (mmol/L)	2.452	2.406		2.506		2.803
	Differences from control (%)	-1.9		2.2		14.3
	Historical control data	1.47 – 2.82
Sodium (mmol/L)	138.7	139.2		139.6		139.5
	Differences from control (%)	0.4		0.7		0.6
	Historical control data	141.6 – 150.4
Bile acids (umol/L)	16.553	17.467		19.536		21.984*
	Differences from control (%)	5.5		18		32.8
	Historical control data	8.14 – 35.55
HDL (mmol/L)	0.937	0.931		1.04		1.301**
	Differences from control (%)	-0.6		11		38.8
	Historical control data	NA
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Summary tables are given in Appendix 1.4.2., individual data in Appendix 2.4.2.

 

Urinalysis

Table 11:Selected urinalysis parameters in males

Urinalysis (males)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Urine volume	16.6	16.75	26.45	51.90^+
	Differences from control (%)	0.9	59.3	212.7
	Historical control data	1.0 - 56.0
pH	7.70	7.70	7.60	7.60
	Differences from control (%)	0	-1.3	-1.3
	Historical control data	6.0 – 8.0
Urinary Leucocytes	0.6	0.6	0.8	0.7
Urine Nitrite	0.1	0.2	0.6	0.7^+
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Table 12:Selected urinalysis parameters in females

Urinalysis (females)	Group / Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Urine volume	12.27	21.60	28.15	22.95
	Differences from control (%)	69.4	120.8	80.0
	Historical control data	1.0 – 42.0
pH	8.00	7.90	8.0	7.3^++
	Differences from control (%)	-1.3	0.0	-8.8
	Historical control data	6.0 – 8.0
Urinary Leucocytes	0.1	0.1	0.1	1.2^++
Urine Nitrite	0.5	0.4	0.4	0.0
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test,              ^+= p<0.05,^++= p<0.01; Dunn two-sided test. Values with significant differences are indicated with bold font.

 

Summary tables are given in Appendix 1.4.3., individual data in Appendix 2.4.3.

 

Organ weights

Table 13:Selected organ weights of the male animals

 

Organ weights (males)	Groups/Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Terminal bodyweight (g)	504.6	515.4	525.4		505.3
	Differences from control (%)	2.1	4.1		0.1
	Historical control data	455 – 657
Liver (g)	14.934	15.643	17.008	17.501^+
	Differences from control (%)	4.7	13.9	17.2
	Historical control data	11.22 – 22.55
Liver / Bodyweight (%)	2.948	3.045	3.237*	3.457**
	Differences from control (%)	3.3	9.8	17.3
	Historical control data	2.183 – 3.442
Liver / Brain weight (%)	660.05	715.06	748.15*	778.48**
	Differences from control (%)	8.3	13.3	17.9
	Historical control data	496.28 – 939.58
Kidney (g)	3.246	3.437	3.779**	4.097**
	Differences from control (%)	5.9	16.4	26.2
	Historical control data	2.32 – 4.22
Kidney / Bodyweight (%)	0.644	0.67	0.721**	0.809**
	Differences from control (%)	4.1	12	25.7
	Historical control data	0.507 – 0.761
Kidney / Brain weight (%)	143.59	157	166.26**	182.37**
	Differences from control (%)	9.3	15.8	27
	Historical control data	103.11 – 189.91
Note: *= p<0.05; **= p<0.01; Dunnett two-sided test. ^+= p<0.05,^++= p<0.01; Dunn two-sided test Values with significant differences are indicated with bold font.

 

 

 

Table 14:Selected organ weights of the female animals

 

Organ weights (females)	Groups/Concentration (mg/kg bw/day)
	Control (0)	Low (100)	Mid (300)		High (1000)
Terminal bodyweight (g)	308.1	315.7	305		302.7
	Differences from control (%)	2.5	-1		-1.8
	Historical control data	254 – 352
Liver (g)	9.244	9.664	10.016	12.842**
	Differences from control (%)	4.5	8.4	38.9
	Historical control data	6.37 – 10.10
Liver / Bodyweight (%)	2.99	3.067	3.287	4.244**
	Differences from control (%)	2.6	9.9	41.9
	Historical control data	2.273 – 3.646
Liver / Brain weight (%)	442.54	456.16	466.74	615.82**
	Differences from control (%)	3.1	5.5	39.2
	Historical control data	307.73 – 561.11
Kidney (g)	2.051	2.192	2.249*	2.309**
	Differences from control (%)	6.9	9.7	12.6
	Historical control data	1.45 – 2.29
Kidney / Bodyweight (%)	0.666	0.695	0.738**	0.765**
	Differences from control (%)	4.4	10.9	14.8
	Historical control data	0.519 – 0.748
Kidney / Brain weight (%)	98.16	103.62	104.75	110.8^++
	Differences from control (%)	5.6	6.7	12.9
	Historical control data	71.43 – 111.67
Note: *= p<0.05; **= p<0.01; Dunnett two-sided test. ^+= p<0.05,^++= p<0.01; Dunn two-sided test Values with significant differences are indicated with bold font.

Summary data are presented in Appendix 1.5.1., individual data in Appendix 2.5.1.

 

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for CEREPLAS DIDS is considered to be 1000 mg/kg bw/day for females. In the males receiving 1000 mg/kg bw/day, histopathological changes were observed in the kidney, which were most probably attributable to Alpha 2u globulin deposition (an adverse nephropathy for male rats only), an effect that is not indicative of a risk for human health. Whilst the rat-specific NOAEL for CEREPLAS DIDS in the males is considered to be 300 mg/kg bw/day, the NOAEL for all the human-relevant endpoints is considered to be 1000 mg/kg bw/day, based on the results of this study.

Executive summary:

In summary, daily administration of CEREPLAS DIDS to Wistar rats during the 90-day treatment period under the conditions of this study did not result in treatment-related mortality or adverse clinical signs.

The bodyweight, body weight gain, and food consumption of the test item treated groups did not show any treatment-related effect. The functional observation battery (FOB) showed no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or treated groups. No treatment-related changes were noted on ophthalmoscopy examination. No clearly adverse treatment-related findings were seen in the clinical pathology parameters. A treatment-related increase of the urine volume was observed in the males receiving 1000 mg/kg bw/day, and the change might be related to the observed microscopic changes. No treatment-related macroscopic findings were detected at necropsy. A dose-dependent increase in the liver and kidney weight was observed in both sexes.

In males receiving 1000 mg/kg bw/day, histopathological changes in the kidney consistent with Alpha 2u globulin nephropathy were observed. In the context of a safety study, this specific finding is a male-rat specific change and is not considered to be adverse for human exposure*. Minimal hepatic weight changes seen in both sexes and hepatic hypertrophy seen in females were considered to be non-adverse, adaptive changes.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for CEREPLAS DIDS is considered to be 1000 mg/kg bw/day for females. In the males receiving 1000 mg/kg bw/day, histopathological changes were observed in the kidney, which were most probably attributable to Alpha 2u globulin deposition (an adverse nephropathy for male rats only), an effect that is not indicative of a risk for human health. Whilst the rat-specific NOAEL for CEREPLAS DIDS in the males is considered to be 300 mg/kg bw/day, the NOAEL for all the human-relevant endpoints is considered to be 1000 mg/kg bw/day, based on the results of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No relevant data were available on laboratory animals given repeated oral treatment with DIDS. Limited subacute studies are available on DIDA and DEHS, and a limited chronic study is available on DOS.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

No data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study on a read-across compound, only briefly described in the secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 12 rats were exposed to DEHS at 25 or 250 mg/m3, 4 hrs/day, 5 days/wk for 13 wk.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data in citing source
- Age at study initiation: no data in citing source
- Weight at study initiation: no data in citing source
- Fasting period before study: no data in citing source
- Housing: no data in citing source
- Diet (e.g. ad libitum): no data in citing source
- Water (e.g. ad libitum): no data in citing source
- Acclimation period: no data in citing source

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data in citing source
- Humidity (%): no data in citing source
- Air changes (per hr): no data in citing source
- Photoperiod (hrs dark / hrs light): no data in citing source

IN-LIFE DATES: no data in citing source

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

not specified

Remarks on MMAD:

MMAD / GSD: No data in citing source

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: no data in citing source
- Method of holding animals in test chamber: no data in citing source
- Source and rate of air: no data in citing source
- Method of conditioning air: no data in citing source
- System of generating particulates/aerosols: no data in citing source
- Temperature, humidity, pressure in air chamber: no data in citing source
- Air flow rate: no data in citing source
- Air change rate: no data in citing source
- Method of particle size determination: no data in citing source
- Treatment of exhaust air: no data in citing source

TEST ATMOSPHERE
- Brief description of analytical method used: no data in citing source
- Samples taken from breathing zone: no data in citing source

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data in citing source.

Duration of treatment / exposure:

13 wk

Frequency of treatment:

4 hr/day, 5 days/wk

Remarks:

Doses / Concentrations:
25 or 250 mg/m3
Basis:
no data

No. of animals per sex per dose:

Total of 12 animals of unspecified sex in each of the two groups.

Control animals:

not specified

Details on study design:

- Dose selection rationale: no data in citing source
- Rationale for animal assignment (if not random): no data in citing source
- Rationale for selecting satellite groups: from citing sources, satellite groups don't appear to have been selected
- Post-exposure recovery period in satellite groups: from citing sources, satellite groups don't appear to have been selected
- Section schedule rationale (if not random): no data in citing source

Positive control:

No data in citing source.

Observations and examinations performed and frequency:

Rats were apparently assessed 1, 7 and 13 weeks after exposure, but no further details are given on any of the examinations performed.

Sacrifice and pathology:

GROSS PATHOLOGY: no data in citing source
HISTOPATHOLOGY: no data in citing source

Other examinations:

No data in citing source.

Statistics:

No data in citing source.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

A secondary source, compiled prior to the results of this study being subject to final analysis, stated that "no significant effects appear evident". Presumably there were no severe treatment-related clinical signs of toxicity, and no treatment-related mortality, but no specific details are given. The finalised results of this study may not have been formalised.

Dose descriptor:

NOAEL

Effect level:

ca. 250 mg/m³ air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Apparently no observed adverse effects at highest tested concentration

Critical effects observed:

not specified

Conclusions:

In a study very briefly described in the secondary literature, no adverse effects were evidently observed in rats following inhalation exposure to DEHS for 13 weeks, resulting in a study NOAEC of 250 mg/m3.

Executive summary:

A subchronic inhalation study has been conducted on DEHS, a structurally-related read-across compound for DIDS, and is reported very briefly in the secondary literature.

Groups of 12 rats were treated with DEHS at either 25 or 250 mg/m3 for 4 hours/day, 5 days/week, for 13 weeks. Rats were apparently "assessed" after 1, 7 and 13 weeks of exposure [no details are given on the extent of examination, and presumably more frequent assessments were also performed e.g. for mortality and clinical signs of toxicity].

According to a secondary source, final analysis of the data has not been formalised. However, no significant adverse effects were evidently reported [no further details given], therefore a study NOAEC of 250 mg/m3 can be assumed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

250 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

No relevant data were available on laboratory animals given repeated inhalation treatment with DIDS. Limited subacute and subchronic studies were available on DEHS, and limited occupational data is available for DOS.

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

Inhalation

No human or laboratory animal data are currently available on the repeated dose inhalation toxicity of DIDS.

 

Relevant read-across data:

No deaths occurred when four rats, two guinea pigs, two rabbits and one cat were exposed to DEHS vapours at 400 mg/m³, 7 hr/day, for 10 days (Treon et al. 1955). A subchronic study, reported briefly in the secondary literature, is available on DEHS. Groups of 12 rats were treated for 4 hr/day, 5 days/wk for 13 wk at 25 or 250 mg/m³. Although the final analysis of the data has not apparently been formalised, no significant effects appeared evident [no further details given] (Swift, 1983). In humans, intrapulmonary deposition of DOS was studied in 58 working coalminers. Total deposition was significantly correlated with measurements of airway obstruction. Simple pneumoconiosis was not associated with the degree of aerosol deposition (Love and Muir, 1976).

Dermal

No repeated-dose dermal studies were identified in humans or laboratory animals on DIDS (or DIDA, and DOS/DEHS).

 

References

Love RG and Muir DC (1976). Aerosol deposition and airway obstruction. American Review of Respiratory Disease 114, 891 -897. [Abstract only.]

Moody DE and Reddy JK (1978). Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicology and Applied Pharmacology 45, 497. Toxicology and Applied Pharmacology 45, 497-504.

 

Treon JF, Cappel JW, Cleveland FP, Larson EE, Atchley RW and Denham RT (1955). The toxicity of the products formed by the thermal decomposition of certain organic substances. American Industrial Hygiene Association Quarterly 16, 187-195.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
OECD Guideline 90-day oral toxicity study on DIDS conducted to GLP.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
OECD Guideline 90-day oral toxicity study on DIDS conducted to GLP.

Justification for classification or non-classification

No adverse effects were seen in male or female rats receiving doses up to 300 mg/kg/day in a standard OECD 408 Guideline study on DIDS. This does not warrant classification under CLP Regulation (EC) 1272/2008.