Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Endpoint summary

Description of key information

The substance was found to be non-toxic during a 90 day repeat dose oral exposure study in the rat. A 21 day palatability study was initially conducted to provide information for the selection of dose levels for further repeated dose toxicity studies. Repeat dose studies via the dermal and inhalation routes of exposure were not conducted on the basis of a lack of exposure to the substance.

Key value for chemical safety assessment

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:

9th June 2008 to 18th December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Food and Drug Administration, Redbook 2000, Toxicological Principles for the Safety Assessment of Food Ingredients (November 2003)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008 Date of Signature: 29/01/2009

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Age at study initiation: Approximately six to eight weeks old
- Weight at study initiation: Males: 188 - 236 g, Females: 110 - 149 g
- Fasting period before study: not stated in report
- Housing: The animals were housed in groups of up to four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Harlan Laboratories UK Ltd, Oxon, UK).
- Diet: ad libitum access to a ground diet (Rat and Mouse SQC Ground Diet No. 1 Diet, Special Diets Services Ltd., Witham, Essex, UK).
- Water: ad libitum access to mains drinking water supplied from polycarbonate bottles attached to the cages
- Acclimation period: Nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to achieve target values of 21 +/- 2°C
- Humidity (%): Set to achieve target value of 55% (+/- 15%)
- Air changes (per hr): At least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light followed by 12 hours darkness

IN-LIFE DATES: From: Day 1 to Day 90 for test animals and control animals. Up to Day 119 for recovery animals

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: not applicable as dietary study

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared prior to treatment and then every two weeks during the treatment period i.e. at twice-monthly intervals

- Mixing appropriate amounts with (Type of food): A known amount of test material was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a hobart QE200 mixer. An additional premix was prepared using the Blixer 4 mixture. These pre-mixes were then added to a larger amount of basal laboratory diet and mixed for a further 30 minutes at a constant speed, setting 1 in a Hobart H800 mixer.

- Storage temperature of food: ambient temperature.
(The diet was stored in labelled, double black plastic bags in labelled, covered plastic bins when not in use).
- The dietary admixture was determined to be stable for a period of three weeks.

VEHICLE
- no vehicle used

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of DVS005u in the dietary admixtures was determined by HPLC using an external standard technique.

The dietary admixtures were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 100 ppm. Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 100 ppm.

The standard and sample solutions were analysed by HPLC using the following conditions:
HPKC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Gemini 5µ C18 (50 x 4.6 mm id)
Mobile phase: Acetonitrile
Flow rate: 1.0 ml/min
UV detector wavelength: 225 nm
Injection volume: 25 µl
Retention time; ~ 3, 7 and 17 minutes

The dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for three weeks.

The dietary admixtures were sampled and analysed within four days of preparation.
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Free access to food

Remarks:

Doses / Concentrations:
1000, 10000 and 20000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

20 males and 20 females per dose group.

10 males and 10 females in the recovery control
10 males and 10 females in the recovery high group

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations were chosen based on the results of the range-finding / palatability investigation detailed as a suporting study for this endpoint in the IUCLID dossier.
- Rationale for animal assignment (if not random): not applicable, as animal selection was random
- Rationale for selecting satellite groups: not stated in report
- Post-exposure recovery period in satellite groups: not stated in report
- Section schedule rationale (if not random): not applicable

Positive control:

Not applicable, as no positive control used.

Observations and examinations performed and frequency:

CAGE SIDE AND NEUROBEHAVIOURAL OBSERVATIONS: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter
- Dose groups that were examined: All non-recovery animals. Functional performance tests were also performed on all non-recovery animals during Week 12.
- Battery of functions tested: Behavioural assessment, sensory reactivity, forelimb/hindlimb grip strength, motor activity

BEHAVIOURAL ASSESSMENTS
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed: gait, posture, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation.

MOTOR ACTIVITY: Animals were assessed with an evaluation period of one hour and the time, in seconds, that each animal was active and mobile was recorded.
- Time schedule for examinations: Daily at the same time if day.

FORELIMB/HINDLIMB GRIP STRENGTH: Animals were allowed to grip a distal metal bar and the force needed to break their grip was recorded.
- Repetition: Three consecutive trials for each animal.
- Apparatus: automated grip strength meter.

SENSORY REACTIVITY: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- The following parameters were observed; grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural changes once daily. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to the start of treatment) and at weekly intervals thereafter. bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was recorded for each group at weekly intervals throughout the study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and during Week 12
- Dose groups that were examined: The eyes of ten males and females from each non-recovery group were examined pre-treatment. The eyes from ten males and females from the non-recovery control and high dose groups were examined before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Week 2, Week 7 and at the end of the study (Day 90) from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91 or Day 120 in the case of recovery animals.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: Ten male and ten female non-recovery animals from each test and control group and all animals of the recovery group.
- Parameters examined: Haemoglobin (Hb) erythrocyte count (RBC), Haematocrit (Hct), erythrocyte indices, total leucocyte count (WBC), differential leucocyte count, platelet count (PLT), reticulocyte count (Retic), prothrombin time (CT) and activated partial thromboplastic time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Week 2, Week 7 and at the end of the study (Day 90) from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91 or Day 120 in the case of recovery animals.
- Animals fasted: No
- How many animals: Ten male and ten female non-recovery animals from each test and control group.
- Parameters examined: Urea, glucose, total protein, albumin, albumin/globulin (A/G) ratio, sodium (Na+), potassium (K+), chloride (Cl-), Calcium (Ca++), inorganic phosphorous (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine, total cholesterol, total bilirubin, triglycerides, gamma-glutamyltranspeptidase and sorbitol dehydrogenase

URINALYSIS: Yes
- Time schedule for collection of urine: Ten male and ten female non-recovery test and control group animals during the final week of the treatment period and on all recovery group animals during the final week of the treatment-free period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood - erythrocytes and haemoglobin, reducing substances and appearance

Sacrifice and pathology:

GROSS PATHOLOGY (Organ weights): Yes - The following organs were removed from test animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus and thyroid/parathyroid.

HISTOPATHOLOGY: Yes - samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: Adrenals, thoracic aorta, bone and bone marrow (for the sternum, femur including stifle joint), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides (preserved in Bouin's fluid then transferred to IMS up to 48 hours later), eyes, gross lesions, harderian gland, heart, ileum (including Payer's patches) jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary glands, skeletal muscles, nasal turbinates, oesophagus, ovaries (with fallopian tubes, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus (corpus and cervix, vagina).

Since there were indications of treatment-related bone marrow changes, examination was subsequently extended to include similarly prepared sections of bone marrow from all animals in the other treatment groups.

Additional information regarding histopathology
All tissues from non-recovery control and 20000 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. All macroscopically observed lesions were also processed.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.

Where appropriate, quantitative data were analysed by the Provantis Tables and Statistics Module. For each variable, the most suitable transformation of the data were found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or steel (non-parametric) test to determine significant differences from the control group. finally, if required, pair-wise tests were performed using the student t-test (parametric) or the Mann-Whitney U-test (non-parametric).

Sorbitol dehydrogenase values were not captured directly onto the Provantis data capture system as this facility was not available. The statistical analyses were undertaken using the SPSS statistical program (version 10). ANOVA, incorporating a test for homogeneity of variance was used. Dose response relationships were investigated by linear regression. Where variances were shown to be homogenous, pair-wise comparisons were conducted using Dunnett's test. Where appropriate, data was analysed using non-parametric methods; Kruskal-Wallis, ANOVA and Mann-Witney U test.

Probability values are presented as follows: p <0.01, p < 0.05, p ≤ 0.05 (not significant).

PLEASE SEE FOLLOWING SECTION FOR ADDITIONAL INFORMATION REGARDING STATISTICS

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Findings not considered to represent an adverse health effect. (Please see following section for details on observations)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Findings not considered to represent an adverse health effect. (Please see following section for details on observations)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths. No clinically observable signs of toxicity were detected. Daily clinical observations detected the presence of an abdominal mass for one 20,000 ppm female from day 81 until the end of the treatment. This was accompanied by diuresis on day 89 and day 90 for this animal. Exophthalmia of the right eye was also detected for one male treated with 1000 ppm from day 55 and one female treated with 1000 ppm was recorded with a small right eye from day 55 until the end of the treatment. These findings were considered incidental and were not considered to represent systemic toxicity.

BEHAVIOURAL ASSESSMENTS
No treatment-related effects in behaviour were detected. One male treated with 1000 ppm displayed exophthalmia during Week 9 assessments. This supported the findings from the daily clinical observations, however, this observation was considered incidental in nature and unrelated to treatment.
All remaining inter and intra groups differences in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS
No treatment-related effects were detected for the grip strength and motor activity assessments. Statistically significant reductions in overall activity were detected for males treated with 10,000 and 1000 ppm when compared to controls, and a reduction in final 20% total activity was detected for 1000 ppm males. No such effects were detected at the highest treatment level, and in the absence of any supporting evidence, these reductions were considered to be unrelated to test material toxicity.

Females treated with 20,000 and 10,000 ppm showed statistically significant reductions in hindlimb grip strength. These intergroup differences, however, were confined to one out of three tests performed on each occasion and the small significance achieved was considered to have arisen incidentally and was unrelated to treatment.

SENSORY REACTIVITY ASSESSMENTS
No treatment related effects were detected in the sensory reactivity assessments. All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of this strain and were of no toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
No adverse effects on bodyweight change were detected. Males treated with 20000 and 10000 ppm displayed statistically significant reductions in bodyweight gain during Week 4 (p<0.01). Statistically significant reductions in bodyweight gains were also detected for males from all treatment groups when compared to controls during Week 8 (p<0.05 to p<0.01). Recovery 20000 ppm males showed a statistically significant increase in bodyweight gain during Week 17 (p<0.01) and a statistically significant reduction during Week 18 (p<0.05). These significant differences detected during the study were confined to the male dose groups, and in the absence of convincing reductions throughout the treatment period, these findings were not considered to represent an adverse effect of treatment.

FOOD CONSUMPTION, FOOD EFFICIENCY, AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency was detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
No ocular effects were detected.

HAEMATOLOGY
No treatment-related effects were detected in comparison to controls at the end of the treatment or recovery periods of the study. During Week 2 assessments, males treated with 20,000 and 10,000 ppm showed statistically significant increase in leucocyte counts. An increase in lymphocyte counts was also detected for males treated with 10,000 ppm. The significance in each case was minimal (p<0.05) and in the absence of similar effects of similar effects detected during the remainder of the study, these increases were considered to have arisen incidentally.

Increases in platelet counts were detected in males treated with 20,000 and 10,000 ppm during Week 2 and Week 7 assessments. Although no such effect was detected during the final week of treatment, an increase in clotting times was detected for males treated with 20,000 ppm during this period. In the absence of any supporting data to suggest a treatment-related effect on the haematopoietic system, these findings were considered to be unrelated to test material toxicity.

Male cell haemoglobin concentration was reduced for males treated with 20,000 ppm during Week 7. Recovery 20,000 ppm females showed a statistically significant increase in MCHC during the final week of the treatment-free period. These findings were of minimal significance (p<0.05) and in the absence of any supporting data, these findings were considered to have arisen incidentally.

BLOOD CHEMISTRY
Aspartate aminotransferase (AP) levels for animals of either sex treated with 20,000 and 10,000 ppm were elevated during the treatment period and the increase was still evident following the recovery period. During Week 2 animals showed a statistically significant increase when compared to the controls (p<0.01). Similar effects were evident during Week 7 and Week 13. Following the treatment free period, a statistically significant increase in AP was still evident for recovery 20,000 ppm males. No effects were detected for animals treated with 1000 ppm.

Plasma cholesterol levels were significantly elevated for females treated with 20,000 and 10,000 ppm during the Week 2 assessments (p<0.05) and was still evident for females treated with 20,000 ppm during Week 7. A similar effect was however not detected prior to the termination of treatment during Week 13, however, during the treatment free phase, recovery 20,000 ppm animals showed a slight but statistically significant reduction in cholesterol. In the absence of any histopathological correlations, these intergroup differences in cholesterol were considered to be unrelated to the test material toxicology.

Males treated with 20,000 or 1000 ppm displayed statistically significant reductions in glucose levels and males form all treated groups displayed a reduction in alanine aminotransferase levels when compared to controls during Week 2. Furthermore, females treated with 20,000 ppm showed an increase in blood calcium levels when compared to controls. The significance achieved in each case was minimal and in the absence of similar effects detected during the Week 7 or Week 13 assessments, these findings were considered to have arisen incidentally.

The remaining statistically significant intergroup differences were isolated in nature and in the absence of any supporting changes, were not considered to be related to test material toxicity.

URINALYSIS
No treatment-related effects were detected. Statistical analysis of the quantitative data did not reveal any significant differences. Red coloured urine with the presence of protein, bilirubin and haemoglobin was evident from the female treated with 20,000 ppm which displayed the abdominal mass and diuresis during the daily clinical observations. Sediment analysis also revealed a higher number of leucocytes and erythrocyte for this animal. These findings were confined to this animal and were considered to be attributable to the abdominal mass detected.

NEUROBEHAVIOUR
No treatment-related effects were detected in the functional performance tests, and the sensory reactivity and behavioural assessments.

ORGAN WEIGHTS
Females treated with 20,000 ppm showed an statistically significant increase in liver and spleen weights, both absolute and relative to terminal weights, when compared to controls. Following the treatment free period, a reduction in liver weights was detected for recovery 20,000 ppm females when compared to recovery controls (p<0.05). No such effects were detected for males treated with 20,000 ppm or for animals of either sex treated with 10,000 or 1000 ppm. Recovery 20,000 ppm females showed a statistically significant increase in thyroid weights. Uterus weights were also elevated when compared to recovery controls. In the absence of similar effects detected following the treatment period and in the absence of any histopathological correlates, these findings are not considered to represent an adverse health effect.

GROSS PATHOLOGY / NECROPSY
No treatment-related macroscopic abnormalities were detected. An abdominal mass, enlarged spleen and red fluid-filled urinary bladder were recorded for the 20,000 ppm female which displayed an abdominal mass and diuresis during the daily clinical observations in the later stages of the study. These were incidental findings considered to have arisen incidentally from a congenital defect in this animal and unrelated to test material toxicity. The remaining findings detected in some control and treated animals were also considered to have risen incidentally. In the absence of any histopathological correlates, these findings were considered to be unrelated to treatment.

HISTOPATHOLOGY
Histopathological examinations did not reveal any treatment-related effects. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed. Although group differences in the incidence or severity of the lesions occasionally attained statistical significance, none was considered to be related to treatment.

Dose descriptor:

NOAEL

Effect level:

1 531 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects - minor treatment related changes at 20,000 and 10,000 ppm which were not considered to represent an adverse health effect.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were detected at the lowest dietary concentration.

Critical effects observed:

not specified

Conclusions:

Oral administration of the test material to rats for a period of ninety consecutive days at dietary concentrations of 20,000, 10,000 and 1000 ppm resulted in minor treatment-related changes at 20,000 and 10,000 ppm. These findings were not considered to represent an adverse health effect, therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 20,000 ppm. No treatment-related effects were detected at the lowest dietary concentration, therefore the 'No Observed Effect Level' (NOEL) was considered to be 1000 ppm.

Executive summary:

Introduction: The study was designed to investigate the systemic toxicity of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day study" (adopted 21 September 1998) and reflects the requirements of the US Food and Drug Administration, Redbook 2000, Toxicological Principles for the Safety Assessment of Food Ingredients (November 2003).

Methods: The test material was administered by dietary admixture to three groups, each of twenty male and twenty female Wistar HAN(TM): HsdRccHan (TM):WIST strain rats, for ninety consecutive days, at dietary concentrations of 1000, 10,000 and 20,000 ppm (equivalent to a mean achieved dosage of 70, 759 and 1531 mg/kg/day respectively). A further group of twenty males and twenty females was exposed to basal laboratory diet to serve as a control. Two recovery groups, each of ten males and ten females, were treated with the high dose (20000 ppm0 or basal laboratory diet for ninety consecutive days and then maintained without treatment for a further twenty-nine days on basal laboratory diet.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for ten males and ten females from each non-recovery dose group during Week 2, Week 7 and at the end of the treatment phase. These investigations were also performed on recovery control and high dose group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on ten males and ten females from each non-recovery dose group, prior to the start of treatment and for ten male and ten female non-recovery control and high dose animals during Week 12 of the study. Urianalytical investigations were undertaken for ten males and ten females from each non-recovery dose group during the final week of treatment and for all recovery group animals during the final week of the treatment-free period. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

Results

Mortality: There were no unscheduled deaths.

Clinical observations: No clinically observable signs of toxicity were detected.

Behavioural assessment: No treatment-related effects were detected.

Functional performance tests: No treatment-related effects were detected.

Sensory reactivity assessments: No treatment-related effects were detected.

Bodyweight: No adverse effect on bodyweight changes was detected.

Food consumption: No adverse effect on dietary intake or food efficiency was detected.

Water consumption: No intergroup differences were detected.

Opthalmoscopy: No intergroup differences were detected.

Urinalysis: No treatment-related effects were detected.

Haematology: No treatment-related effects were detected.

Blood chemistry: Aspartate aminotransferase levels for animals treated with 20000 and 10000 ppm were higher than controls during the treatment period and the increase was still evident for recovery 20000 ppm males following the treatment-free period. No such effects were detected for animals of either sex treated with 1000 ppm.

Organ weights: Females treated with 20000 ppm showed an increase in liver and spleen weights, both absolute and relative to terminal weights, when compared to controls. no such effects were evident following the treatment free period. No such effects were detected for males treated with 20000 ppm or for animals of either sex treated with 10000 or 1000 ppm.

Necropsy: No treatment-related macroscopic abnormalities were detected.

Histopathology: No treatment-related changes were detected.

Conclusion: Oral administration of the test material DVS005u, to rats for a period of ninety consecutive days at dietary concentrations of 20,000, 10,000 and 1000 ppm resulted in minor treatment-related changes at 20,000 and 10,000 ppm. these findings were not considered to represent an adverse health effect, therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 20,000 ppm. No treatment-related effects were detected at the lowest dietary concentration, therefore the 'No Observed Effect Level' (NOEL) was considered to be 1000 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 531 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Two studies, both with Klimisch scores of 1, are available, therefore the quality of database is acceptable.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Introduction

The study was designed to investigate the systemic toxicity of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day study" (adopted 21 September 1998) and reflects the requirements of the US Food and Drug Administration, Redbook 2000, Toxicological Principles for the Safety Assessment of Food Ingredients (November 2003).

Methods

The test material was administered by dietary admixture to three groups, each of twenty male and twenty female Wistar HAN(TM): HsdRccHan (TM):WIST strain rats, for ninety consecutive days, at dietary concentrations of 1000, 10000 and 20000 ppm (equivalent to a mean achieved dosage of 70, 759 and 1531 mg/kg/day respectively). A further group of twenty males and twenty females was exposed to basal laboratory diet to serve as a control. Two recovery groups, each of ten males and ten females, were treated with the high dose (20000 ppm) or basal laboratory diet for ninety consecutive days and then maintained without treatment for a further twenty-nine days on basal laboratory diet.

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for ten males and ten females from each non-recovery dose group during Week 2, Week 7 and at the end of the treatment phase. These investigations were also performed on recovery control and high dose group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on ten males and ten females from each non-recovery dose group, prior to the start of treatment and for ten male and ten female non-recovery control and high dose animals during Week 12 of the study. Urianalytical investigations were undertaken for ten males and ten females from each non-recovery dose group during the final week of treatment and for all recovery group animals during the final week of the treatment-free period. All animals were subjected to gross necropsy examination and a comprehensive histopathological evaluation of tissues was performed.

Results

Mortality:There were no unscheduled deaths.

Clinical observations: No clinically observable signs of toxicity were detected.

Behavioural assessment:No treatment-related effects were detected.

Functional performance tests: No treatment-related effects were detected.

Sensory reactivity assessments: No treatment-related effects were detected.

Bodyweight: No adverse effect on bodyweight changes was detected.

Food consumption: No adverse effect on dietary intake or food efficiency was detected.

Water consumption: No intergroup differences were detected.

Opthalmoscopy: No intergroup differences were detected.

Urinalysis: No treatment-related effects were detected.

Haematology: No treatment-related effects were detected.

Blood chemistry: Aspartate aminotransferase levels for animals treated with 20000 and 10000 ppm were higher than controls during the treatment period and the increase was still evident for recovery 20000 ppm males following the treatment-free period. No such effects were detected for animals of either sex treated with 1000 ppm.

Organ weights: Females treated with 20000 ppm showed an increase in liver and spleen weights, both absolute and relative to terminal weights, when compared to controls. no such effects were evident following the treatment free period. No such effects were detected for males treated with 20000 ppm or for animals of either sex treated with 10000 or 1000 ppm.

Necropsy: No treatment-related macroscopic abnormalities were detected.

Histopathology: No treatment-related changes were detected.

Conclusion

Oral administration of the test material DVS005u, to rats for a period of ninety consecutive days at dietary concentrations of 20000, 10000 and 1000 ppm resulted in minor treatment-related changes at 20000 and 10000 ppm. These findings were not considered to represent an adverse health effect, therefore the No Observed Adverse Effect Level (NOAEL) was considered to be 20000 ppm.

This study was ranked as Reliability 1 according to the Klimisch scale, as it was conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Dermal

A repeat dose dermal toxicity study was not conducted on the substance as dermal contact during formulation and use is unlikely, and physicochemical and toxicological properties do not suggest a significant rate of absorption through the skin.

Inhalation

Testing by the inhalation route of exposure is considered inappropriate as exposure to humans via inhalation is considered unlikely, as there is no possibility of exposure to aerosols, particles or droplets of an inhalable size.

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

90 day study considered to be the key study with the 28 day study considered to be the supporting study on the basis that the study conducted over the longest time frame should be the key study.

Justification for classification or non-classification

The substance did not meet the criteria for classification as toxic or harmful by the oral route of exposure (NOAEL of 1531 mg/kg/day) during a 90 day repeat dose toxicity study in the rat.