Reaction mass of 2-[2-(benzoyloxy)ethoxy]ethyl benzoate, 1-[2-(benzoyloxy)propoxy]propan-2-yl benzoate, and 2-[2-[2-(benzoyloxy)ethoxy]ethoxy]ethyl benzoate

Endpoint summary

Description of key information

No specific testing was performed on the reaction mass. However, 13-week repeated dose oral dose (dietary) studies on components of the reaction mass were performed to OECD guidelines and in compliance with GLP to determine the effects of prolonged exposure on rats of the test materials. Groups of ten rats were dosed by dietary administration with DPGDB or DEGDB for a period of 13 weeks at levels 0 (untreated diet control), 250, 1000, 1750, and 2500 mg/kgbw/day. The dose levels for TEGDB were 0 (untreated diet control), 400, 1000, 1600 and 2200 mg/kgbw/day. DPGDB, DEGDB and TEGDB were found to be non-toxic orally under the conditions of these repeated dose toxicity tests. The NOAEL was determined at 1000mg/kg/day in each case.

The NOAEL for the reaction mass is also 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 1997 - 10 September 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD and Japanese test guidelines, and in compliance with GLP.

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: Toxicity test guidelines published in Notification Yakushin 1 No. 24 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare, dated 11 September 1989.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:(IGS) CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles Rives Breeding Laboratories, Manston Road, Margate, Kent, UK.
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 231 to 301 g for males, 161 to 213 g for females.
- Housing: Housed in suspended cages with wire mesh floors, 5 rats of the same sex per cage. Each cage measured 25.7 cm high, 35.8 cm wide, and 53 cm in depth.
- Diet (e.g. ad libitum): Free access to ground SDS Rat and Mouse No.1 maintenance diet
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: 19 days from receipt of animals until allocation to groups; a further 7 days from allocation until the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21 ± 2°C; actual range 16 - 28°C
- Humidity (%): Nominally 55 ± 10%; actual range 42 - 80%
- Photoperiod (hrs dark / hrs light): 12 hours darkness / 12 hours light per 24 hour period.

IN-LIFE DATES: From: 16 April 1997 (Animal arrival) / 12 May 1997 (First day of dosing) To: 14 August 1997 (Main Kill) or 10 September 1997 (Recovery kill)

Route of administration:

oral: feed

Vehicle:

other: SDS Rat and Mouse No. 1 maintenance diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): SDS Rat and Mouse No. 1 maintenance diet
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At weeks 1 and 11 a representative sample of the dietary formulation at each dose level was obtained, sub-sampled, extracted (soxhlet extraction in acetone, then diluted in acetone as appropriate, then evaporated to dryness using RFE, and re-dissolved in HPLC mobile phase), then analysed by HPLC-UV.
Prior to the first analysis, the stability and homogeneity of the formulations was confirmed at nominal concentrations 50 ppm and 60000 ppm for up to 22 days at ambient temperature. The analytical method was validated before use with respect to linearity of detector response, precision of injection, specificity, limit of detection, and accuracy and precision of the extraction technique (procedural recovery).
Mean concentrations for DEGDB in test diet formulations analysed during the study were within 7% of nominal concentrations, confirming the accuracy of formulation.

Duration of treatment / exposure:

13 weeks. Selected animals in the control and high dosage levels were maintained for a 4 week recovery period to monitor the reversibility of any treatment-related effects.

Frequency of treatment:

Continuous (the test material was administered in the diet, which was available ad liitum)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations: 250, 1000, 1750, 2500 mg/kg/day
The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations: 250, 1000, 1750, 2500 mg/kg/day
The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

1 750 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations: 250, 1000, 1750, 2500 mg/kg/day
The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations: 250, 1000, 1750, 2500 mg/kg/day
The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

No. of animals per sex per dose:

10, 20 for control and high level groups to account for recovery animals.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Treatment levels were selected by the Sponsor on the basis of available toxicity data specifically a preliminary toxicity study performed at this laboratory.
- Rationale for animal assignment (if not random): Not applicable - random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Animals were observed for behavioural changes, reaction to treatment, or signs of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to groups, then once a week thereafter.

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

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: No - a group mean value for the food convertion ratio was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored visually daily throughout the study. Water consumption was measured accurately, by weight, over daily periods during week 12 of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examined before commencement of daose, and during week 13 of dosing.
- Dose groups that were examined: All rats examined before dosing; all rats in groups 1 and 5 (control and 2500 mg/kg/day) examined at week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Total white blood cell count, Differential white blood cell count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Cell morphology, Platelet count, Reticulocyte count, Prothrombin time, Activated partial thromboplastin time, and Thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Total protein, Glucose, Urea nitrogen, Creatinine, Alkaline phosphatase, Glutamic-pyruvic transaminase, Glutamic-oxolacetic transaminase, Gamma-glutamyltransferase, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorous, Cholesterol, Protein electrphoresis (Albumin, α1-Globulin, α2-Globulin, β-Globulin, γ-Globulin, Total globulin), A/G ratio, Ornithine carbamoyl transferase, and Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Volume, pH, Specific grvity, Protein, Urinary sodium, Urinary potassium, Urinary chloride, Total reducing substances, Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments, and Microscopy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (See table 1, below)
HISTOPATHOLOGY: Yes

Statistics:

The following sequence of statistical tests was used for food consumption water consumption bodyweight clinical pathology and organ weight data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%) the proportion of animals with values different from the mode was analysed (Fisher 1950 and Mantel 1963).
Otherwise:
A test was applied to test for heterogeneity of variance between treatments (Bartlett 1937). Where significant (at the 1% level) heterogeneity was found a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected or if a satisfactory transformation was found a one way analysis of variance was carried out.
If significant heterogeneity of variance was present and could not be removed by a transformation an analysis of ranks was used (Kruskal and Wallis 1952/3).
Analyses of variance were followed by Student's t test and Williams test (1971/2) for a dose related response although only the one thought most appropriate for the response pattern observed was reported. The Kruskal Wallis analyses were followed by the non parametric equivalents of these tests (Shirley 1977).
Where appropriate analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level in an attempt to allow for differences in bodyweight which may have influenced the organ weights.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male receiving 2500 mg/kg/day experienced 2 convulsive like episodes and was in poor condition thereafter. This animal was sacrificedi nWeek 4. Aniother male receiving 2500 mg/kg/day showed a similar episode on a single occasion but fully recovered. A hunched posture and body tremors were noted during the treatment period principally in rats receiving 2500 mg/kg/day. These findings were considered to be related to the stress associated with treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male receiving 2500 mg/kg/day was sacrificed in Week 4 due to poor clinical condition following 2 convulsive like episodes

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gain fluctuated in all treated groups but was reduced to a toxicologically significant degree only at 1750 or 2500 mg/kg/day. An adverse palatability reaction in Weeks 1 to 2 was insufficient to fully account for these changes. Weight gain for animals previously receiving 2500 mg/kg day was vastly superior to that of Controls in the Recovery period such that the intergroup deficit was almost offset within 4 weeks.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There appeared to be an adverse palatibility reaction over the first few days of treatment, with all treated groups spilling a significant quantity of diet from the food hoppers. The amount of spillage appeared to be approximately dosage-related. Towards the end of the first week the amount of spillage decreased. Group mean food consumption by males receiving 1750 or 2500 mg/kg/day was notably reduced in Week I and slightly reduced in Week 2 (with a dosage relationship). Females receiving 2500 mg/kg/day showed a marginally lower consumption in Week 1 only. In subsequent weeks, food consumption by treated groups was considered to be normal and overall consumption to Week 13 and throughout the Recovery period was essentially comparable to that of the Controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The efficiency of food (utilisation assessed by calculation of the food conversion ratios (FCRs)) was below that of Controls for both sexes receiving 2500 mg/kg/day throughout the treatment period and for both sexes receiving 1750 mg/kg/day during the latter half of the treatment period. During the Recovery period the efficiency of food utilisation for both sexes previously treated at 2500 mg/kg/day was vastly superior to that of the controls reflecting the improved bodyweight performance at that time.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The minor intergroup differences in mean erythrocyte parameters some of which achieved statistical significance were considered of no toxicological importance in the absence of a dosage relationship and/or consistency between the sexes and as the majority of results were characteristic for rats of this age. The only notable individual finding was an apparent anaemia in two rats at 2500 mg/kg/day, characterised by low PCV haemoglobin erythrocyte count and mean corpuscular haemoglobin concentrations together with a high reticulocyte count indicative of a regenerative anaemia. This is considered related to treatment and indicative of inadequate compensatory erythropoiesis in a small number of susceptible rats.
All individual white cell data and the results of clotting tests (PT, TT and APTT) were normal for rats of this age and strain therefore the minor intergroup differences are considered to be coincidental and of no toxicological importance. A single animal at 2500 mg/kg/day showed a very low platelet count at Week 13 which slightly influenced the group mean result. In isolation this single low result cannot be attributed to treatment.
There were no other notable findings at Week 13.
By Week 4 of the Recovery period the regenerative anaemia previously noted in the two rats at 2500 mg/kg/day had fully recovered. All other group mean and individual values were considered to be characteristic for rats of this age despite the few statistically significant intergroup differences.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma glucose levels at Week 13 were towards or below the lower end of the nonnal range in a number of females at all dosage levels and in males receiving 1750 or 2500 mg/kg/day, leading to a degree of statistical significance for males receiving 1750 mg/kg/day and both sexes treated with 2500 mg/kg/day. Intergroup differences were however small and only three animals at 2500 mg/kg/day showed remarkably low individual results.
In Recovery Week 4 several individuals previously receiving 2500 mg/kg/day continued to exhibit low plasma glucose levels and although the group mean result was below that of the Controls to a statistically significant degree the actual group mean result had improved slightly from that recorded at Week 13, indicating a general trend for recovery.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The urine of treated animals showed a dosage related increase in acidity and sodium potassium concentrations were reduced in both sexes receiving 1750 or 2500 mg/kg/day which may be associated with excretion of acidic metabolites.

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weight as a percentage of terminal bodyweight was notably increased for a number of animals of both sexes receiving 2500 mg/kg/day at the Main kill. A corresponding increase in group mean values adjusted for, and as a percentage of, terminal bodyweight was noted for both sexes receiving 2500 mg/kg/day and generally achieved a level of statistical significance. Upon completion of the Recovery period, the intergroup differences in liver weight were small and considered to be indicative of complete recovery. The increased liver weight at the highest doses correlated with the observation ofminimal liver cell hypertrophy.
Although group mean spleen weight adjusted for terminal bodyweight was increased to a statistically significant degree for females receiving 1000 mg/kg/day or above this finding cannot be conclusively attributed to treatment particularly as there was no similar trend in absolute spleen weight in male treated groups and as the histopathological findings in the spleen could not wholly account for the change in the weight of this organ at the Main kill.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Liver - Periportal hepatocyte hypertrophy (minimal in degree) was seen in the majority of males and an occasional female rat receiving 2500 mg/kg/day. This finding is not inconsistent with the minimal increase in individual and group mean liver weights adjusted and as a percentage of terminal bodyweight for males and females receiving 2500 mg/kg/day and the increased values recorded for liver transaminases for male rats receiving 2500 mg/kg/day.
Spleen - Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However an increased incidence and degree of haemosiderosis was seen in male and female rats receiving 2500 and 1750 mg/kg/day. This was considered to be a treatment related exacerbation of this physiological change.

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
One male receiving 2500 mg/kg/day was sacrificed in Week 4 due to poor clinical condition following 2 convulsive like episodes. Another male receiving 2500 mg/kg/day showed a similar episode on a single occasion but fully recovered. A hunched posture and body tremors were noted during the treatment period principally in rats receiving 2500 mg/kg/day. These findings were considered to be related to the stress associated with treatment.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain fluctuated in all treated groups but was reduced to a toxicologically significant degree only at 1750 or 2500 mg/kg/day. An adverse palatability reaction in Weeks 1 to 2 was insufficient to fully account for these changes. Weight gain for animals previously receiving 2500 mg/kg day was vastly superior to that of Controls in the Recovery period such that the intergroup deficit was almost offset within 4 weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There appeared to be an adverse palatibility reaction over the first few days of treatment, with all treated groups spilling a significant quantity of diet from the food hoppers. The amount of spillage appeared to be approximately dosage-related. Towards the end of the first week the amount of spillage decreased. Group mean food consumption by males receiving 1750 or 2500 mg/kg/day was notably reduced in Week I and slightly reduced in Week 2 (with a dosage relationship). Females receiving 2500 mg/kg/day showed a marginally lower consumption in Week 1 only. In subsequent weeks, food consumption by treated groups was considered to be normal and overall consumption to Week 13 and throughout the Recovery period was essentially comparable to that of the Controls.

FOOD EFFICIENCY
The efficiency of food (utilisation assessed by calculation of the food conversion ratios (FCRs)) was below that of Controls for both sexes receiving 2500 mg/kg/day throughout the treatment period and for both sexes receiving 1750 mg/kg/day during the latter half of the treatment period. During the Recovery period the efficiency of food utilisation for both sexes previously treated at 2500 mg/kg/day was vastly superior to that of the controls reflecting the improved bodyweight performance at that time.

HAEMATOLOGY
The minor intergroup differences in mean erythrocyte parameters some of which achieved statistical significance were considered of no toxicological importance in the absence of a dosage relationship and/or consistency between the sexes and as the majority of results were characteristic for rats of this age. The only notable individual finding was an apparent anaemia in two rats at 2500 mg/kg/day, characterised by low PCV haemoglobin erythrocyte count and mean corpuscular haemoglobin concentrations together with a high reticulocyte count indicative of a regenerative anaemia. This is considered related to treatment and indicative of inadequate compensatory erythropoiesis in a small number of susceptible rats.
All individual white cell data and the results of clotting tests (PT, TT and APTT) were normal for rats of this age and strain therefore the minor intergroup differences are considered to be coincidental and of no toxicological importance. A single animal at 2500 mg/kg/day showed a very low platelet count at Week 13 which slightly influenced the group mean result. In isolation this single low result cannot be attributed to treatment.
There were no other notable findings at Week 13.
By Week 4 of the Recovery period the regenerative anaemia previously noted in the two rats at 2500 mg/kg/day had fully recovered. All other group mean and individual values were considered to be characteristic for rats of this age despite the few statistically significant intergroup differences.

CLINICAL CHEMISTRY
Plasma glucose levels at Week 13 were towards or below the lower end of the nonnal range in a number of females at all dosage levels and in males receiving 1750 or 2500 mg/kg/day, leading to a degree of statistical significance for males receiving 1750 mg/kg/day and both sexes treated with 2500 mg/kg/day. Intergroup differences were however small and only three animals at 2500 mg/kg/day showed remarkably low individual results.
In Recovery Week 4 several individuals previously receiving 2500 mg/kg/day continued to exhibit low plasma glucose levels and although the group mean result was below that of the Controls to a statistically significant degree the actual group mean result had improved slightly from that recorded at Week 13, indicating a general trend for recovery.

URINALYSIS
The urine of treated animals showed a dosage related increase in acidity and sodium potassium concentrations were reduced in both sexes receiving 1750 or 2500 mg/kg/day which may be associated with excretion of acidic metabolites.

ORGAN WEIGHTS
Liver weight as a percentage of terminal bodyweight was notably increased for a number of animals of both sexes receiving 2500 mg/kg/day at the Main kill. A corresponding increase in group mean values adjusted for, and as a percentage of, terminal bodyweight was noted for both sexes receiving 2500 mg/kg/day and generally achieved a level of statistical significance. Upon completion of the Recovery period, the intergroup differences in liver weight were small and considered to be indicative of complete recovery. The increased liver weight at the highest doses correlated with the observation ofminimal liver cell hypertrophy.
Although group mean spleen weight adjusted for terminal bodyweight was increased to a statistically significant degree for females receiving 1000 mg/kg/day or above this finding cannot be conclusively attributed to treatment particularly as there was no similar trend in absolute spleen weight in male treated groups and as the histopathological findings in the spleen could not wholly account for the change in the weight of this organ at the Main kill.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver - Periportal hepatocyte hypertrophy (minimal in degree) was seen in the majority of males and an occasional female rat receiving 2500 mg/kg/day. This finding is not inconsistent with the minimal increase in individual and group mean liver weights adjusted and as a percentage of terminal bodyweight for males and females receiving 2500 mg/kg/day and the increased values recorded for liver transaminases for male rats receiving 2500 mg/kg/day.
Spleen - Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However an increased incidence and degree of haemosiderosis was seen in male and female rats receiving 2500 and 1750 mg/kg/day. This was considered to be a treatment related exacerbation of this physiological change.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Conclusions:

There were no findings of toxicological importance at a dosage of 1000 mg/kgbw/day or below. When selected animals previously receiving 2500 mg/kgbw/day were maintained off-dose for 4 weeks, all treatment-related changes showed evidence of, or complete recovery.

Executive summary:

A 13 -week repeated oral dose (dietary) study was conducted to determine the effects of prolonged exposure on rats of the test material DEGDB. The study was conducted according to OECD and Japanese test guidelines, and in compliance with GLP.

Groups of five rats (of each sex) were dosed by dietary administration with DEGDB for a period of 13 weeks at levels 0 (untreated diet control), 250, 1000, 1750, and 2500 mg/kgbw/day. Additional rats were dosed at 0 and 2500 mg/kgbw/day to allow for an assessment of recovery from treatment for four weeks after dosing. No findings of toxicological importance were detected in this study at a dosage of 1000 mg/kgbw/day or below. Dosages of 1750 or 2500 mg/kgbw/day were tolerated (with one exception - a single mortality at 2500 mg/kg/day) but induced clinical findings changes in blood parameters, minor treatment-related pathology and/or adverse effects on bodyweight gain. When selected animals previously receiving 2500 mg/kgbw/day were maintained off dose for 4 weeks all treatment related changes showed evidence of or complete recovery. The NOEL was 1000 mg/kgbw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 1997 - 10 September 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD and Japanese test guidelines and in compliance with GLP.

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: Toxicity test guidelines published in Notification Yakushin 1 No. 24 of the Pharmaceutical Affairs Bureau, Japanese Ministry of Health and Welfare, dated 11 September 1989.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: (IGS) CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston Road, Margate, Kent, UK
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 220 to 297 g for males and 159 to 208 g for females.
- Housing: Housed in suspended cages with wire mesh floors, 5 rats of the same sex per cage. Each cage measures 25.7 cm high, 35.8 cm wide and 53 cm in depth.
- Diet: Free access to ground SDS Rat and Mouse No 1 maintenance diet
- Water: Free access to tap water
- Acclimation period: 19 days from receipt of animals until allocation to groups; a further 7 days from allocation until the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21 ± 2°C, actual range 17.5-28°C
- Humidity (%): Nominally 55 ± 10%;, actual range 38-74%
- Photoperiod: 12 hours darkness / 12 hrs light per 24 hour period

IN-LIFE DATES: From: 16 April 1997 (Animal arrival) / 12 May 1997 (First day of dosing) To: 11- 14 August 1997 (Main Kill) or 9 - 10 September 1997 (Recovery kill)

Route of administration:

oral: feed

Vehicle:

other: SDS Rat and Mouse No. 1 maintenance diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): SDS Rat and Mouse No. 1 maintenance diet
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At weeks 1 and 11 a representative sample of the dietary formulation at each dose level was obtained, sub-sampled, extracted (soxhlet extraction in acetone, then diluted in acetone as appropriate, then evaporated to dryness using RFE, and re-dissolved in HPLC mobile phase), then analysed by HPLC-UV.
Prior to the first analysis, the stability and homogeneity of the formulations was confirmed at nominal concentrations 50 ppm and 60000 ppm for up to 22 days at ambient temperature. The analytical method was validated before use with respect to linearity of detector response, precision of injection, specificity, limit of detection, and accuracy and precision of the extraction technique (procedural recovery).
Mean concentrations for DPGDB in test diet formulations analysed during the study were within 10% of nominal concentrations, confirming the accuracy of formulation.

Duration of treatment / exposure:

13 weeks. Selected animals in the control and high dosage levels were maintained for a 4 week recovery period to monitor the reversibility of any treatment-related effects.

Frequency of treatment:

Continuous (the test material was administered in the diet, which was available ad liitum)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

(The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

(The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.)

Dose / conc.:

1 750 mg/kg bw/day (nominal)

Remarks:

(The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.)

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

(The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.)

No. of animals per sex per dose:

10, 20 for control and high level groups to account for recovery animals.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Treatment levels were selected by the Sponsor on the basis of available toxicity data specifically a preliminary toxicity study performed.
- Rationale for animal assignment (if not random): Not applicable - random

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Animals were observed for behavioural changes, reaction to treatment, or signs of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to groups, then once a week thereafter.

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

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: No - a group mean value for the food convertion ratio was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored visually daily throughout the study. Water consumption was measured accurately, by weight, over daily periods during week 12 of the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examined before commencement of dose, and during week 13 of dosing.
- Dose groups that were examined: All rats examined before dosing; all rats in groups 1 and 5 (control and 2500 mg/kg/day) examined at week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Total white blood cell count, Differential white blood cell count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Cell morphology, Platelet count, Reticulocyte count, Prothrombin time, Activated partial thromboplastin time, and Thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4.
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: Total protein, Glucose, Urea nitrogen, Creatinine, Alkaline phosphatase, Glutamic-pyruvic transaminase, Glutamic-oxolacetic transaminase, Gamma-glutamyltransferase, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorous, Cholesterol, Protein electrphoresis (Albumin, α1-Globulin, α2-Globulin, β-Globulin, γ-Globulin, Total globulin), A/G ratio, Ornithine carbamoyl transferase, and Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: Samples taken from all animals in week 13, and for all recovery animals in recovery week 4
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: Volume, pH, Specific grvity, Protein, Urinary sodium, Urinary potassium, Urinary chloride, Total reducing substances, Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments, and Microscopy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1, below)
HISTOPATHOLOGY: Yes

Statistics:

The following sequence of statistical tests was used for food consumption, water consumption, bodyweight, clinical pathology and organ weight data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%) the proportion of animals with values different from the mode was analysed (Fisher 1950 and Mantel 1963). Otherwise:
A test was applied to test for heterogeneity of variance between treatments (Bartlett 1937). Where significant (at the 1% level) heterogeneity was found a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected or ifa satisfactory transformation was found a one way analysis of variance was carried out If significant heterogeneity of variance was present and could not be removed by a transformation an analysis of ranks was used (Kruskal and Wallis 1952/3).
Analyses of variance were followed by Student's t test and Williams test (1971/2) for a dose related response although only the one thought most appropriate for the response pattern observed was reported. The Kruskal Wallis analyses were followed by the non parametric equivalents of these tests (Shirley 1977).
Where appropriate analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level in an attempt to allow for differences in bodyweight which may have influenced the organ weights.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no findings which could be conclusively related to treatment. Incidental findings of hairloss and stained fur were noted, however the incidence showed no correlation to dosage and/or these findings were also noted in the Control group.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a general dosage-related adverse effect on group mean bodyweight gain in both sexes. With a few exceptions (females receiving 250 mg/kg/day in Week 1, males treated with 1000 mg/kg/day Weeks 6 to 13) this was generally evident throughout the dosing period. However in rats treated with 1000 mg/kg/day or below the degree of change was insufficient to be of toxicological importance. This is supported by the fact that final bodyweights for males and females at 1000 mg/kg/day were only 5% less than controls.
The degree of suppression of weight gain (-14%) in females receiving 1000 mg/kg/day exceeded that of males at this dosage (-8%) and achieved statistical significance. However this finding is considered unlikely to be of toxicological importance as data from higher dose groups indicate that males were more likely to be susceptible to treatment-related adverse effect on bodyweight.
Small reductions in food intake were insufficient to fully account for the adverse effect on bodyweight.
During the 4-week Recovery period, animals previously receiving 2500 mg/kg/day showed vastly superior weight gain than Controls, however 4 weeks appeared to be an insufficient time to completely rectify the effects of 13 weeks treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

An overall slight but dosage related reduction in group mean food consumption was noted for males receiving 1750 or 2500 mg/kg/day. In addition a slight reduction in group mean food intake was noted for females receiving 2500 mg/kg/day principally during Week 1 of treatment. The quantities of spilt diet recorded indicated that there may have been a slight adverse palatability reaction in these groups particularly during Week 1. Consumption by all other groups and by rats previously receiving 2500 mg/kg/day during the Recovery period was considered to be comparable to that of Controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

As treatment-related effects bodyweight were more pronounced than those on food consumption, trends in the efficiency of food utilisation generally reflected the bodyweight change, namely;
With a few exceptions there was a general dosage-related adverse effect of treatment on the efficiency of food utilisation in both sexes. The degree of change was however small at the lower dosages and was considered to be of toxicological importance only in both sexes receiving 1750 or 2500 mg/kg/day.
During the Recovery period the efficiency of food utilisation for both sexes previously treated at 2500 mg/kg/day was superior to that of the controls, reflecting the improved bodyweight performance at that time.

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no changes at Week 13 considered to be related to treatment. All findings were characteristic of the age and strain of animals employed. Ophthalmoscopy was therefore not performed during the Recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Several parameters at Week 13 achieved levels of statistical significance as detailed below, however none could be conclusively related to treatment.
A number of erythrocyte parameters achieved levels of statistical significance in Week 13 for rats receiving 1000 mg/kg/day or above (increased red cell counts, reduced mean corpuscular volume and reduced mean corpuscular haemoglobin). There were, however, few indications of a dosage-relationship and most individual values were either within the range encountered in Controls or were generally characteristic of rats of this age (within the range of background data at this laboratory). These minor intergroup differences are therefore considered not to be of toxicological importance.
Similarly, levels of statistical significance were achieved for clotting tests (PT, IT and APTT) in one or both sexes at 1000 mg/kg/day or above. The only indication of a dosage relationship was for a reduction in male TT and APTT data, but with the exception of a low TT for Rats 43M (1750 mg/kg/day)and 60M (2500 mg/kg/day), all individual values were characteristic of this species (and the group mean data for treated females generally exceeded that of the Controls). Intergroup differences in clotting tests are therefore considered to be coincidental.
Individual leukocyte data for males receiving 2500 mg/kg/day at Week 13 were generally towards the lower end of the normal range. Although statistical significance was achieved for group mean values, the lowest single result at Week 13 was for a Control animal (Rat 135M). In addition, group meanleukocyte data of females receiving 2500 mg/kg/day generally exceeded that of Controls. The slightly lower group mean results for males therefore cannot be conclusively attributed to treatment.
There were no other notable findings at Week 13 and all data at Recovery Week 4 was unremarkable.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma AP activity was very high at Week 13 for 4 rats receiving 2500 mg/kg/day and slightly raised for 6 males receiving 2500 mg/kg/day and 3 rats treated with 1750 mg/kg/day. Levels of statistical significance were achieved for male group mean values at 1750 or 2500 mg/kg/day. By Recovery Week 4 all AP activities of surviving animals had returned to a level comparable to that of Controls.
There was no similar effect of treatment in females. All individual female AP activities were characteristic of the species and those in treated groups were generally comparable to the range encountered in the Controls. The level of statistical significance achieved for female group mean values at Week 13 is therefore coincidental. Female untreated rats are more prone to exhibit spontaneously raised plasma GPT, GOT and OCT, activities than their male counterparts. In addition rats only slightly older than those employed in this study sometimes show sporadic and unexplained high transaminase activities.
In Recovery Week 4 the only notable plasma enzyme activities were in Rat 117F previously receiving 2500 mg/kg/day In isolation and as spontaneous high individual activities are not unknown this finding is considered likely to be coincidental. All other animals previously receiving 2500 mg/kg/day had enzyme activities comparable to concurrent Controls indicating complete reversibility of the treatment related changes. Group mean plasma cholesterol was increased to a statistically significant degree in males receiving 1000 mg/kg/day or above but without a dosage relationship. The group mean cholesterol level of females treated with 2500 mg/kg/day was slightly in excess of Controls and achieved statistical significance however in the absence of a dosage relationship this was considered not to be of toxicological importance In Recovery Week 4 all cholesterol levels were considered normal.
No other findings were considered to be of toxicological importance

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The following treatment related findings were noted, however not all individuals were affected. In the absence of renal pathology these are considered to be of little toxicological importance and may represent alteration in renal function and/or be related to excretion of the test material or its metabolites:
Group mean urinary volume was reduced and specific gravity increased at Week 13 for males receiving 1750 or 2500 mg/kg/day with a dosage-relationship however there was no indication of a similar effect in females.
There was a dosage-related effect upon group mean urinary pH of both sexes receiving 1000 mg/kg/day or above at Week 13 with the urine of treated animals being increasingly acidic at higher dosages levels.
Group mean urinary sodium and potassium concentrations were reduced in Week 13 for both sexes treated with 1750 or 2500 mg/kg/day with results generally achieving statistical significance and showing a dosage relationship in these groups.
By Recovery Week 4, results for animals previously treated with 2500 mg/kg/day were considered comparable to Controls.
There were no other notable findings. Intergroup differences in urinary protein and chloride levels were considered of no toxicological importance as all individual results were unremarkable.

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a slight treatment related increase in liver weight for females receiving 1750 mg/kg/day and males treated with 2500 mg/kg/day. Females receiving 2500 mg/kg/day showed a more marked increase in liver weight. These changes may be partially associated with increased plasma transaminase levels and the low grade hepatic hypertrophy detected microscopically, however, there was little consisitency between these data, the degree of hepatic toxicity is considered to be low.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver - Periportal hepatocyte hypertrophy (dose related) was seen in the males and female rats receiving 1750 or 2500 mg/kg/day. This finding is associated with the increased group mean liver weight adjusted for bodyweight recorded for male and female rats receiving 2500 mg/kg/day and females receiving 1750 mg/kg/day. This microscopic finding was also associated with the increased group mean values recorded for liver transaminases in this treatment group. The Recovery group animals demonstrated that this was a reversible effect.
Spleen - Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However the slight degree of haemosiderosis seen in both male and female rats receiving 2500 and in a smal number of female rats receiving 1750 mg/kg/day was considered to be a treatment related exacerbation of this physiological change. The Recovery group animals demonstrated that this was a reversible finding.
Caecum - An increased incidence of minimal epithelial hyperplasia was seen in male and female rats receiving 2500 mg/kg/day compared to controls. The Recovery group animals demonstrated that this was a reversible effect.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths. There were no findings which could be conclusively related to treatment. Incidental findings of hairloss and stained fur were noted, however the incidence showed no correlation to dosage and/or these findings were also noted in the Control group.

BODY WEIGHT AND WEIGHT GAIN
There was a general dosage-related adverse effect on group mean bodyweight gain in both sexes. With a few exceptions (females receiving 250 mg/kg/day in Week 1, males treated with 1000 mg/kg/day Weeks 6 to 13) this was generally evident throughout the dosing period. However in rats treated with 1000 mg/kg/day or below the degree of change was insufficient to be of toxicological importance. This is supported by the fact that final bodyweights for males and females at 1000 mg/kg/day were only 5% less than controls.
The degree of suppression of weight gain (-14%) in females receiving 1000 mg/kg/day exceeded that of males at this dosage (-8%) and achieved statistical significance. However this finding is considered unlikely to be of toxicological importance as data from higher dose groups indicate that males were more likely to be susceptible to treatment-related adverse effect on bodyweight.
Small reductions in food intake were insufficient to fully account for the adverse effect on bodyweight.
During the 4-week Recovery period, animals previously receiving 2500 mg/kg/day showed vastly superior weight gain than Controls, however 4 weeks appeared to be an insufficient time to completely rectify the effects of 13 weeks treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
An overall slight but dosage related reduction in group mean food consumption was noted for males receiving 1750 or 2500 mg/kg/day. In addition a slight reduction in group mean food intake was noted for females receiving 2500 mg/kg/day principally during Week 1 of treatment. The quantities of spilt diet recorded indicated that there may have been a slight adverse palatability reaction in these groups particularly during Week 1. Consumption by all other groups and by rats previously receiving 2500 mg/kg/day during the Recovery period was considered to be comparable to that of Controls.

FOOD EFFICIENCY
As treatment-related effects bodyweight were more pronounced than those on food consumption, trends in the efficiency of food utilisation generally reflected the bodyweight change, namely;
With a few exceptions there was a general dosage-related adverse effect of treatment on the efficiency of food utilisation in both sexes. The degree of change was however small at the lower dosages and was considered to be of toxicological importance only in both sexes receiving 1750 or 2500 mg/kg/day.
During the Recovery period the efficiency of food utilisation for both sexes previously treated at 2500 mg/kg/day was superior to that of the controls, reflecting the improved bodyweight performance at that time.

OPHTHALMOSCOPIC EXAMINATION
There were no changes at Week 13 considered to be related to treatment. All findings were characteristic of the age and strain of animals employed. Ophthalmoscopy was therefore not performed during the Recovery period.

HAEMATOLOGY
Several parameters at Week 13 achieved levels of statistical significance as detailed below, however none could be conclusively related to treatment.
A number of erythrocyte parameters achieved levels of statistical significance in Week 13 for rats receiving 1000 mg/kg/day or above (increased red cell counts, reduced mean corpuscular volume and reduced mean corpuscular haemoglobin). There were, however, few indications of a dosage-relationship and most individual values were either within the range encountered in Controls or were generally characteristic of rats of this age (within the range of background data at this laboratory). These minor intergroup differences are therefore considered not to be of toxicological importance.
Similarly, levels of statistical significance were achieved for clotting tests (PT, IT and APTT) in one or both sexes at 1000 mg/kg/day or above. The only indication of a dosage relationship was for a reduction in male TT and APTT data, but with the exception of a low TT for Rats 43M (1750 mg/kg/day)and 60M (2500 mg/kg/day), all individual values were characteristic of this species (and the group mean data for treated females generally exceeded that of the Controls). Intergroup differences in clotting tests are therefore considered to be coincidental.
Individual leukocyte data for males receiving 2500 mg/kg/day at Week 13 were generally towards the lower end ofthe normal range. Although statistical significance was achieved for group mean values, the lowest single result at Week 13 was for a Control animal (Rat 135M). In addition, group meanleukocyte data of females receiving 2500 mg/kg/day generally exceeded that of Controls. The slightly lower group mean results for males therefore cannot be conclusively attributed to treatment.
There were no other notable findings at Week 13 and all data at Recovery Week 4 was unremarkable.

CLINICAL CHEMISTRY
Plasma AP activity was very high at Week 13 for 4 rats receiving 2500 mg/kg/day and slightly raised for 6 males receiving 2500 mg/kg/day and 3 ratstreated with 1750 mg/kg/day. Levels of statistical significance were achieved for male group mean values at 1750 or 2500 mg/kg/day. By Recovery Week 4 all AP activities of surviving animals had returned to a level comparable to that of Controls.
There was no similar effect of treatment in females. All individual female AP activities were characteristic of the species and those in treated groups were generally comparable to the range encountered in the Controls. The level of statistical significance achieved for female group mean values at Week 13 is therefore coincidental. Female untreated rats are more prone to exhibit spontaneously raised plasma GPT, GOT and OCT, activities than their male counterparts. In addition rats only slightly older than those employed in this study sometimes show sporadic and unexplained high transaminase activities.
In Recovery Week 4 the only notable plasma enzyme activities were in Rat 117F previously receiving 2500 mg/kg/day In isolation and as spontaneous high individual activities are not unknown this finding is considered likely to be coincidental. All other animals previously receiving 2500 mg/kg/day had enzyme activities comparable to concurrent Controls indicating complete reversibility of the treatment related changes. Group mean plasma cholesterol was increased to a statistically significant degree in males receiving 1000 mg/kg/day or above but without a dosage relationship. The group mean cholesterol level of females treated with 2500 mg/kg/day was slightly in excess of Controls and achieved statistical significance however in the absence of a dosage relationship this was considered not to be of toxicological importance In Recovery Week 4 all cholesterol levels were considered normal.
No other findings were considered to be of toxicological importance

URINALYSIS
The following treatment related findings were noted, however not all individuals were affected. In the absence of renal pathology these are considered to be of little toxicological importance and may represent alteration in renal function and/or be related to excretion of the test material or its metabolites:
Group mean urinary volume was reduced and specific gravity increased at Week 13 for males receiving 1750 or 2500 mg/kg/day with a dosage-relationship however there was no indication of a similar effect in females.
There was a dosage-related effect upon group mean urinary pH of both sexes receiving 1000 mg/kg/day or above at Week 13 with the urine of treated animals being increasingly acidic at higher dosages levels.
Group mean urinary sodium and potassium concentrations were reduced in Week 13 for both sexes treated with 1750 or 2500 mg/kg/day with results generally achieving statistical significance and showing a dosage relationship in these groups.
By Recovery Week 4, results for animals previously treated with 2500 mg/kg/day were considered comparable to Controls.
There were no other notable findings. Intergroup differences in urinary protein and chloride levels were considered of no toxicological importance as all individual results were unremarkable.

ORGAN WEIGHTS
There was a slight treatment related increase in liver weight for females receiving 1750 mg/kg/day and males treated with 2500 mg/kg/day. Females receiving 2500 mg/kg/day showed a more marked increase in liver weight. These changes may be partially associated with increased plasma transaminase levels and the low grade hepatic hypertrophy detected microscopically however there was little consisitency between these data, the degree of hepatic toxicity is considered to be low.

HISTOPATHOLOGY: NON-NEOPLASTIC
Liver - Periportal hepatocyte hypertrophy (dose related) was seen in the males and female rats receiving 1750 or 2500 mg/kg/day. This finding is associated with the increased group mean liver weight adjusted for bodyweight recorded for male and female rats receiving 2500 mg/kg/day and females receiving 1750 mg/kg/day. This microscopic finding was also associated with the increased group mean values recorded for liver transaminases in this treatment group. The Recovery group animals demonstrated that this was a reversible effect.
Spleen - Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However the slight degree of haemosiderosis seen in both male and female rats receiving 2500 and in a smal number of female rats receiving 1750 mg/kg/day was considered to be a treatment related exacerbation of this physiological change. The Recovery group animals demonstrated that this was a reversible finding.
Caecum - An increased incidence of minimal epithelial hyperplasia was seen in male and female rats receiving 2500 mg/kg/day compared to controls. The Recovery group animals demonstrated that this was a reversible effect.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Conclusions:

1000 mg/kg/day is considered to represent a non-toxic dose in both sexes. Dosages of 1750 or 2500 mg/kg/day were tolerated but induced changes in blood parameters, minor treatment related pathology and/or adverse effects on bodyweight gain. When selected animals previously receiving 2500 mg/kg/day were maintained off-dose for 4 weeks all treatment related changes showed evidence of, or complete recovery.

Executive summary:

A 13 -week repeated oral dose (dietary) study was conducted to determine the effects of prolonged exposure to rats of the test material DPGDB. The study was conducted according to OECD and Japanese test guidelines, and in compliance with GLP.

Groups of five rats (of each sex) were dosed by dietary administration with DEGDB for a period of 13 weeks at levels 0 (untreated diet control), 250, 1000, 1750, and 2500 mg/kgbw/day. Additional rats were dosed at 0 and 2500 mg/kgbw/day to allow for an assessment of recovery from treatment 4 weeks after the end of dosing.

Clinical observations showed no signs of adverse affects with the exception of a decrease in bodyweight gain and final bodyweight in both sexes at the higher doses. The major toxicological findings were limited to the liver, spleen and caecum at the 1750 and/or 2500 mg/kg/day doses. The liver showed minimal to slight liver cell hypertrophy (enlargement) and alterations in associated biochemical parameters. At 1000 mg/kgbw/day there was a slight increase in liver enzymes but not judged to be sufficient for toxicity. The spleen showed a slight to moderate increase in the normal degree of haemosiderosis (iron accumulation) and the caecum showed a minimal epithelial hyperplasia only at 2500 mg/kg/day. Urinary pH was decreased in a dose related manner in both sexes which was likely due to acidic metabolites being excreted in the urine and may have been related to the decreased elimination of sodium and potassium at the 1750 and 2500 mg/kg/day doses only. Most importantly all treatment related affects were reversible or showed tendency to reverse in the 2500 mg/kgbw/day day 4 week recovery group.

No findings of toxicological importance were detected in this study at a dosage of 1000 mg/kgbw/day or below. The NOAEL was 1000 mg/kgbw/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 1997 - 14 January 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD, EPA and Japanese test guidelines, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Toxicity test guidelines published in Notification Yakushin 1 No 24 of the Pharmaceutical Affairs Bureau Japanese Ministry of Health and Welfare dated11 September 1989

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:(IGS) CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston Road, Margate Kent, UK.
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 219 to 264g for males; 153 to 205g for females.
- Housing: Housed in suspended cages with wire mesh floors, 5 rats of the same sex per cage. Each cage measured 25.7 cm high, 35.8 cm wide and 53 cm in depth
- Diet (e.g. ad libitum): Free access to ground SDS Rat and Mouse No 1 maintenance diet
- Wate (e.g. ad libitum): Free access to tap water
- Acclimation period: 14 days from receipt of animals until allocation to groups; a further 7 days from allocation until the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21 ± 2°C, actual range 16 - 26°C
- Humidity (%): Nominally 55 ± 10%, actual range 38 - 74%
- Photoperiod (hrs dark / hrs light): 12 hours darkness / 12 hours light per 24 hour period

IN-LIFE DATES: From: 27 August 1997 (Animal arrival) / 17 September 1997 (First day of dosing) To: 18 December 1997 (Main kill) 14 January 1998 (Recovery kill)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly.
- Mixing appropriate amounts with (Type of food): TEGDB was warmed gently up to 40°C in order to induce a liquid state prior to dietary formulation. A pre-mix was prepared each week by stirring the test substance directly into SDS Rat and Mouse No 1 maintenance diet until diet was dry enough to sieve. The mixture was then passed through a 1 mm sieve and made up to the required weight before being mixed in a Turbula mixer for a period of 5 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet, homogeneity being achieved by mixing in a Turbula mixer for a minimal period of 5 minutes using the relevant standard operating procedures.
- Storage temperature of food: Room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At weeks 1, 11 and 13 a representative sample of the dietary formulation at each dose level was obtained, sub-sampled, extracted (soxhlet extraction in acetone, then diluted in acetone as appropriate, then evaporated to dryness using RFE, and re-dissolved in HPLC mobile phase), then analysed by HPLC-UV.
Prior to the first analysis, the stability and homogeneity of the formulations was confirmed at nominal concentrations 500 ppm and 60000 ppm for up to 22 days at ambient temperature. The analytical method was validated before use with respect to linearity of detector response, precision of injection, specificity, limit of detection, and accuracy and precision of the extraction technique (procedural recovery).
Mean concentrations for TEGDB in test diet formulations analysed during the study were within 7% of nominal concentrations, confirming the accuracy of formulation.

Duration of treatment / exposure:

13 weeks. Selected animals in the control and high dosage levels were maintained for a 4 week recovery period to monitor the reversibility of any treatment-related effects.

Frequency of treatment:

Continuous (the test material was administered in the diet, which was available ad liitum)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Basis: other: The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: other: The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

1 600 mg/kg bw/day (nominal)

Remarks:

Basis: other: The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

Dose / conc.:

2 200 mg/kg bw/day (nominal)

Remarks:

Basis: other: The concentrations of test substance in diet were changed each week to preserve the required dosage levels for each group.

No. of animals per sex per dose:

10, 20 for control and high level groups to account for recovery animals.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Treatment levels were selected by the Sponsor on the basis of available toxicity data specifically a preliminary toxicity study performed at this laboratory
- Rationale for animal assignment (if not random): Not applicable - random

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Animals were observed for behavioural changes, reaction to treatment, or signs of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to groups, on the day of commencemnt of treatment, then once a week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg bodyweight/day: No - food comsumption per cage and per group was calculateded for each week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No - a group mean value for the food convertion ratio was calculated.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Monitored visually daily throughout the study. Water consumption was measured accurately, by weight, over daily periods during week 12 of the study. As there was no effect of treatment further investigations were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examined before commencement of dosing, and during week 13 of dosing.
- Dose groups that were examined: All rats examined before dosing; all rats in groups 1 and 5 (control and 2200 mg/kg/day) examined at week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples taken from all animals during weeks 5, 13 and from recovery animals in recovery week 4.
- Anaesthetic used for blood collection: Yes (under isofluorane nitrous oxide anaesthesia)
- Animals fasted: Yes overnight prior to sampling
- How many animals: All animals
- Parameters checked: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Total white blood cell count, Differential white blood cell count, Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Cell morphology, Platelet count, Reticulocyte count, Prothrombin time, Activated partial thromboplastin time, and Thrombotest.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples taken from all animals during weeks 5, 13 and from recovery animals in recovery week 4.
- Animals fasted: Yes overnight prior to sampling
- How many animals: All animals
- Parameters checked: Glucose, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase (Glutamic-pyruvic transaminase), Asparate aminotransferase (Glutamic-oxolacetic transaminase), Gamma-glutamyltransferase, Sodium, Potassium, Calcium, Chloride, Inorganic phosphorous, Cholesterol, Protein electrphoresis (Albumin, α1-Globulin, α2-Globulin, β-Globulin, γ-Globulin, Total globulin), A/G ratio, Ornithine carbamoyl transferase, and Triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight urine samples were collected from all animals in week 13 and for recovery animals in recovery week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes and water removed overnight
- Parameters checked: Volume, pH, Specific grvity, Protein, Urinary sodium, Urinary potassium, Urinary chloride, Total reducing substances, Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments, and Microscopy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. (See table 1, below)
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females.
The following sequence of statistical tests was used for food consumption, water consumption, bodyweight, clinical pathology and organ weight data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%) the proportion of animals with values different from the mode was analysed (Fisher 1950 and Mantel 1963).
Otherwise:
A test was applied to test for heterogeneity of variance between treatments (Bartlett 1937). Where significant (at the 1 % level) heterogeneity was found a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
If no significant heterogeneity was detected (or if a satisfactory transformation was found) a one way analysis of variance was carried out If significant heterogeneity of variance was present and could not be removed by a transformation an analysis of ranks was used (Kruskal and Wallis 1952/3).
Analyses of variance were followed by Student's t test and Williams test (1971/2) for a dose-related response although only the one thought most appropriate for the response pattern observed was reported. The Kruskal Wallis analyses were followed by the non parametric equivalents of these tests (Shirley 1977).
Where appropriate analysis ofcovariance was used in place of analysis of variance in the above sequence. For organ weight data analysis of variance was performed using terminal bodyweight as covariate when the within group relationship between organ weight and bodyweight was significant at the 10% level in an attempt to allow for differences in bodyweight which may have influenced the organ weights. Organ weight data was presented relative to both terminal bodyweight and brain weight.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the treatment period, hair loss was apparent in 6/20 males and 3/20 females at 2200 mg/kg/day in comparison with 0/20 males and 4/20 female control animals. During the recovery period hair loss was apparent in 2/9 males and 6/10 females at 2200 mg/kg/day in comparison with 1/10 male and 3/0 female control animals. The finding was first noted from Week 15 for the male animals and from Week 4 for 1 female, Week 12 for 2 of the female animals and from Week 15 for the remaining females. There were no other clinical signs considered to be related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death of a male at 2200 mg/kg/day, which was sacrificed due to poor condition in Week 12. Microscopic pathology revealed the factors contributory to death were lymphoblastic/lymphocytic lymphoma. This death was not associated with treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweights were decreased in both sexes at the two highest dose levels 1600 and 2200 mg/kg/day. This was more apparent in males than females.
At 1000 mg/kg/day and lower dose levels, no biologically significant bodyweight decrease was observed. This was determined by final total bodyweights and by bodyweight gain. While females showed a statistically significant lower bodyweight gain at Week 13 given 1000 mg/kg/day and above, there was no dose response exhibited nor was there any dose response observed for final total bodyweights. Moreover no female dose level exceeded an 8% decrease for final bodyweights. Therefore, this indicates that a 1000 mg/kg/day dose is the minimal no effect level for bodyweight changes.
During the recovery period the male and female animals at 2200 mg/kg/day showed a statistically significant increase in bodyweight gain indicating recovery from the treatment period. The four week recovery period for females suggests that any bodyweight loss was recoverable during this time. Week 4 should not be included in this portion of the recovery analysis because the animals were being tested and disturbed during this final recovery week. For males, the recovery period showed definitive signs of recovery of bodyweight. However since the males had a greater differential bodyweight gain to return to control weights than females, they did not fully regain all the weight lost during the treatment period. Nonetheless both sexes showed a significant recovery in bodyweights when treatment was terminated.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for the first week of treatment for males at 2200 mg/kg/day was slightly lower than controls, the value attaining statistical significance.
During the same period females at 400, 1600 and 2200 mg/kg/day also showed a lower food intake when compared with controls but with no effect at 1000 mg/kg/day and no dose relationship apparent this is thought to be fortuitous. Food consumption for both males and females during weeks 2 to 13 was generally comparable for all treated groups.
During weeks 1 to 13 a slightly lower food consumption was noted for males at 2200 mg/kg/day, in comparison with controls this value attaining statistical significance. During the same period both sexes at 400 mg/kg/day demonstrated a decreased food consumption though these values did not attain statistical significance all other treated groups were generally comparable with control values and consequently no relationship to treatment is suspected. During the recovery period the food consumption for both males and females at 2200 mg/kg/day was generally comparable with their respective control groups.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

For the period, weeks 1-13, male and female animals at 1600 and 2200 mg/kg/day and females only at 1000 mg/kg/day showed an inferior efficiency of food utilisation compared with controls. No dose relationship was apparent in the females. For the recovery period, male and female animals at 2200 mg/kg/day showed a superior efficiency of food utilisation in comparison with the control animals reflecting the improved bodyweight performance at this time.

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

no effects observed

Description (incidence and severity):

Analysis of the week 13 water consumption revealed that the intakes for each treated group were comparable with their respective control groups and that there was no effect of treatment. As a result water consumption was not assessed during the recovery period.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Week 13 ophthalmic investigation revealed no changes that were attributable to treatment. All findings were characteristic of the age and strain of animals employed. Ophthalmoscopy was therefore not performed during the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When measured at week 5 in males only at 1600 and 2200 mg/kg/day dose groups, red blood cell (RBC) indicators suggested an increase in RBC's.
However at week 13, the same parameters were not consistently increased in males. Neither did the females show the same trend with the same parameters at either week 5 or 13.
Similarly, males showed a non cellular specific decrease in white blood cells (WBC) at the two highest dose levels (1600 and 2200 mg/kg/day) but the females showed no indications whatsoever of a decrease in WBC s at any dose level.
An integrated interpretation might suggest a decrease in RBC parameters with the excess haemosiderin in the spleen suggesting a breakdown of circulating RBC's. However, if this were the case, the reticulocyte count would be elevated indicating synthesis of new RBC's. This is not noted in either sex at any dose level. While the increased RBC' s in males may or may not be treatment related the toxicological significance remains obscure. Moreover, no biologically significant alterations werenoted for any haematological parameters at the 1000mg/kg/day level or below.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of the week 5 (Day 32), week 13 and week 17 (recovery) biochemistry investigation revealed a number of statistically significant differences between the control and treated groups but all of these were slight with no clear shifts apparent in the range of the individual values or consistency between the weeks and sexes.
The parameters showing the clearest shift in range ofindividual values from controls were:
In week 5, very slight increases in ALT and AST were noted in both sexes at 2200 mg/kg/day plus females at 1600 mg/kg/day (ALT only) was noted. In week 13, a similar finding was still apparent for the males only at 2200 mg/kg/day and was associated with periportal hepatocyte hypertrophy seen in the majority of males at 2200 mg/kg/day and an occasional female at 2200 mg/kg/day. This difference was not apparent for the week 17 recovery investigation and there was no residual hepatic pathology. In weeks 5, 13 and 17 (recovery), a slight decrease in cholesterol in females at 2200 mg/kg/day was noted; as this finding was not apparent for males at this dose level it is considered not to be of toxicological importance.
In weeks 5 and 13, a slight increase in triglycerides was noted in females at 2200 mg/kg/day. This change was not apparent at the Week 17 (recovery) investigation and was not seen in male animals at this dose level It is considered not to be of toxicological importance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In week 13, analysis revealed reduced urinary sodium, potassium and pH levels for both sexes at 2200 mg/kg/day.
In addition reduced pH levels were noted for males only at 1600 mg/kg/day.
Week 17 (recovery) analysis revealed reduced urinary sodium and pH levels for females at 2200 mg/kg/day. This reduction in pH is considered to be a normal physiological consequence of excretion of acidic metabolites.
Other findings attaining statistical significance were slight in degree with individual values similar to those seen in control animals.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

All organ weights were generally comparable with the concurrent control groups and there were no findings considered of toxicological importance.
Statistical analyses of the organ weights revealed several values attaining statistical significance however the differences were generally slight and inconsistent between the sexes.
The only change showing a degree of consistency was a slight increase in lung weight in both sexes receiving 2200 mg/kg/day and males receiving 1600 mg/kg/day which was not apparent during the recovery kill.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination at the terminal post mortem revealed alopecia in 4/10 animals receiving 2200 mg/kg/day in comparison with 0/10 controls, whilst at the recovery post mortem, alopecia was seen in 2/9 male and 4/10 female animals receiving 2200 mg/kg/day, these findings were related to the in-life clinical observation of hair loss. The incidence and distribution of all other findings was considered to fall within the expected background range of macroscopic changes and was not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes:
Liver: Periportal hepatocyte hypertrophy (minimal in degree) was seen in the majority of males and an occasional female rat receiving 2200 mg/kg/day.
Spleen: Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However, an increased incidence and degree of haemosiderosis was seen in male and female rats receiving 2200 mg/kg/day. This was considered to be a treatment related exacerbation of this physiological change.
Recovery period:
Treatment-related changes:
Liver: No treatment related changes were detected in rats receiving 2200 mg/kg/day after the 4 week recovery period. The periportal hepatocyte hypertrophy seen in the liver of rats in this study at the end of the treatment period were therefore considered to be reversible.
Spleen: The degree of haemosiderosis was still increased in male and female rats receiving 2200 mg/kg/day, although there was evidence of recovery. A slight increased incidence of minimal extramedullary haemopoiesis was seen in male and female rats receiving 2200mg/kg/day after the 4 week recovery period.
Incidental findings: All other findings seen at the end of treatment and after the recovery period were considered to be incidental and of no toxicological importance. In particular, there were no findings to account for the slightly increased lung weight seen in both sexes receiving 2200 mg/kg/day and males receiving 1600 mg/kg/day after the treatment period.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
There was one unscheduled death of a male at 2200 mg/kg/day, which was sacrificed due to poor condition in Week 12. Microscopic pathology revealed the factors contributory to death were lymphoblastic/lymphocytic lymphoma. This death was not associated with treatment.
During the treatment period, hair loss was apparent in 6/20 males and 3/20 females at 2200 mg/kg/day in comparison with 0/20 males and 4/20 female control animals. During the recovery period hair loss was apparent in 2/9 males and 6/10 females at 2200 mg/kg/day in comparison with 1/10 male and 3/0 female control animals. The finding was first noted from Week 15 for the male animals and from Week 4 for 1 female, Week 12 for 2 of the female animals and from Week 15 for the remaining females. There were no other clinical signs considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN:
Bodyweights were decreased in both sexes at the two highest dose levels 1600 and 2200 mg/kg/day. This was more apparent in males than females. At 1000 mg/kg/day and lower dose levels, no biologically significant bodyweight decrease was observed. This was determined by final total bodyweights and by bodyweight gain. While females showed a statistically significant lower bodyweight gain at Week 13 given 1000 mg/kg/day and above, there was no dose response exhibited nor was there any dose response observed for final total bodyweights. Moreover no female dose level exceeded an 8% decrease for final bodyweights. Therefore, this indicates that a 1000 mg/kg/day dose is the minimal no effect level for bodyweight changes.
During the recovery period the male and female animals at 2200 mg/kg/day showed a statistically significant increase in bodyweight gain indicating recovery from the treatment period. The four week recovery period for females suggests that any bodyweight loss was recoverable during this time. Week 4 should not be included in this portion of the recovery analysis because the animals were being tested and disturbed during this final recovery week. For males, the recovery period showed definitive signs of recovery of bodyweight. However since the males had a greater differential bodyweight gain to return to control weights than females, they did not fully regain all the weight lost during the treatment period. Nonetheless both sexes showed a significant recovery in bodyweights when treatment was terminated.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption for the first week of treatment for males at 2200 mg/kg/day was slightly lower than controls, the value attaining statistical significance. During the same period females at 400, 1600 and 2200 mg/kg/day also showed a lower food intake when compared with controls but with no effect at 1000 mg/kg/day and no dose relationship apparent this is thought to be fortuitous. Food consumption for both males and females during weeks 2 to 13 was generally comparable for all treated groups.
During weeks 1 to 13 a slightly lower food consumption was noted for males at 2200 mg/kg/day, in comparison with controls this value attaining statistical significance. During the same period both sexes at 400 mg/kg/day demonstrated a decreased food consumption though these values did not attain statistical significance all other treated groups were generally comparable with control values and consequently no relationship to treatment is suspected. During the recovery period the food consumption for both males and females at 2200 mg/kg/day was generally comparable with their respective control groups.

FOOD EFFICIENCY:
For the period, weeks 1-13, male and female animals at 1600 and 2200 mg/kg/day and females only at 1000 mg/kg/day showed an inferior efficiency of food utilisation compared with controls. No dose relationship was apparent in the females.
For the recovery period, male and female animals at 2200 mg/kg/day showed a superior efficiency of food utilisation in comparison with the control animals reflecting the improved bodyweight performance at this time.

WATER CONSUMPTION:
Analysis of the week 13 water consumption revealed that the intakes for each treated group were comparable with their respective control groups and that there was no effect of treatment. As a result water consumption was not assessed during the recovery period.

OPHTHALMOSCOPIC EXAMINATION:
Week 13 ophthalmic investigation revealed no changes that were attributable to treatment. All findings were characteristic of the age and strain of animals employed. Ophthalmoscopy was therefore not performed during the recovery period.

HAEMATOLOGY:
When measured at week 5 in males only at 1600 and 2200 mg/kg/day dose groups, red blood cell (RBC) indicators suggested an increase in RBC's.
However at week 13, the same parameters were not consistently increased in males. Neither did the females show the same trend with the same parameters at either week 5 or 13.
Similarly, males showed a non cellular specific decrease in white blood cells (WBC) at the two highest dose levels (1600 and 2200 mg/kg/day) but the females showed no indications whatsoever of a decrease in WBC s at any dose level.
An integrated interpretation might suggest a decrease in RBC parameters with the excess haemosiderin in the spleen suggesting a breakdown of circulating RBC's. However, if this were the case, the reticulocyte count would be elevated indicating synthesis of new RBC's. This is not noted in either sex at any dose level. While the increased RBC' s in males may or may not be treatment related the toxicological significance remains obscure. Moreover, no biologically significant alterations werenoted for any haematological parameters at the 1000mg/kg/day level or below.

CLINICAL CHEMISTRY:
Analysis of the week 5 (Day 32), week 13 and week 17 (recovery) biochemistry investigation revealed a number of statistically significant differences between the control and treated groups but all of these were slight with no clear shifts apparent in the range of the individual values or consistency between the weeks and sexes.
The parameters showing the clearest shift in range ofindividual values from controls were:
In week 5, very slight increases in ALT and AST were noted in both sexes at 2200 mg/kg/day plus females at 1600 mg/kg/day (ALT only) was noted. In week 13, a similar finding was still apparent for the males only at 2200 mg/kg/day and was associated with periportal hepatocyte hypertrophy seen in the majority of males at 2200 mg/kg/day and an occasional female at 2200 mg/kg/day. This difference was not apparent for the week 17 recovery investigation and there was no residual hepatic pathology.
In weeks 5, 13 and 17 (recovery), a slight decrease in cholesterol in females at 2200 mg/kg/day was noted; as this finding was not apparent for males at this dose level it is considered not to be of toxicological importance.
In weeks 5 and 13, a slight increase in triglycerides was noted in females at 2200 mg/kg/day. This change was not apparent at the Week 17 (recovery) investigation and was not seen in male animals at this dose level It is considered not to be of toxicological importance.

URINALYSIS:
In week 13, analysis revealed reduced urinary sodium, potassium and pH levels for both sexes at 2200 mg/kg/day. In addition reduced pH levels were noted for males only at 1600 mg/kg/day. Week 17 (recovery) analysis revealed reduced urinary sodium and pH levels for females at 2200 mg/kg/day. This reduction in pH is considered to be a normal physiological consequence of excretion of acidic metabolites.
Other findings attaining statistical significance were slight in degree with individual values similar to those seen in control animals.

ORGAN WEIGHTS:
All organ weights were generally comparable with the concurrent control groups and there were no findings considered of toxicological importance. Statistical analyses of the organ weights revealed several values attaining statistical significance however the differences were generally slight and inconsistent between the sexes. The only change showing a degree of consistency was a slight increase in lung weight in both sexes receiving 2200 mg/kg/day and males receiving 1600 mg/kg/day which was not apparent during the recovery kill.

GROSS PATHOLOGY:
The macroscopic examination at the terminal post mortem revealed alopecia in 4/10 animals receiving 2200 mg/kg/day in comparison with 0/10 controls, whilst at the recovery post mortem, alopecia was seen in 2/9 male and 4/10 female animals receiving 2200 mg/kg/day, these findings were related to the in-life clinical observation of hair loss. The incidence and distribution of all other findings was considered to fall within the expected background range of macroscopic changes and was not related to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Treatment-related changes:
Liver: Periportal hepatocyte hypertrophy (minimal in degree) was seen in the majority of males and an occasional female rat receiving 2200 mg/kg/day.
Spleen: Haemosiderosis is a normal physiological response to red cell turnover in rats and is most frequently present to a minimal degree in control rats. However, an increased incidence and degree of haemosiderosis was seen in male and female rats receiving 2200 mg/kg/day. This was considered to be a treatment related exacerbation of this physiological change.
Recovery period:
Treatment-related changes:
Liver: No treatment related changes were detected in rats receiving 2200 mg/kg/day after the 4 week recovery period. The periportal hepatocyte hypertrophy seen in the liver of rats in this study at the end of the treatment period were therefore considered to be reversible.
Spleen: The degree of haemosiderosis was still increased in male and female rats receiving 2200 mg/kg/day, although there was evidence of recovery. A slight increased incidence of minimal extramedullary haemopoiesis was seen in male and female rats receiving 2200mg/kg/day after the 4 week recovery period.
Incidental findings: All other findings seen at the end of treatment and after the recovery period were considered to be incidental and of no toxicological importance. In particular, there were no findings to account for the slightly increased lung weight seen in both sexes receiving 2200 mg/kg/day and males receiving 1600 mg/kg/day after the treatment period.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

not specified

Lower bodyweight gains were apparent for both sexes receiving 1600 or 2200 mg/kg/day. A slight reduction in food intake was noted for high level males. Dietary administration of Benzoflex S-358 at 2200 mg/kg/day correlated with pathological changes in the liver and spleen. Changes in or associated with the liver included an increase in plasma enzyme activities and periportal hepatocyte hypertrophy. Changes in the spleen were increased incidence and degree of haemosiderosis.

During a 4 week recovery period, bodyweight gain for animals previously receiving 2200 mg/kg/day was superior to that of the controls such that the intergroup deficit was almost offset. Following recovery liver enzyme activity was normal and there was no residual pathology. The incidence and degree of haemosiderosis was reduced but recovery was not complete.

Conclusions:

No Observed Effect Level (NOEL) was 1000 mg/kg/day. Dosages of 1600 and 2200 mg/kg/day were tolerated over a 13 week period but induced some or all of changes in blood parameters, minor treatment related pathology and adverse effects on bodyweight gain and food consumption. When selected animals previously receiving 2200 mg/kg/day were maintained off-dose for 4 weeks, all treatment related changes showed complete or tended towards recovery.

Executive summary:

A 13 -week repeated oral dose (dietary) study was conducted to determine the effects of prolonged exposure on rats of the test material TEGDB. The study was conducted according to OECD, Japanese and EPA test guidelines, and in complinace with GLP.

Groups of five rats (of each sex) were dosed by dietary administration with TEGDB for a preriod of 13 weeks at levels 0 (intreated diet control), 200, 1000, 1600 and 2200 mg/kgbw/day. Additional rats were dosed at 0 and 2200 mg/kgbw/day to allow for an assessment of recovery from treatment 4 weeks after dosing.

The principal findings during the study were in rats receiving 1600 mg/kg/day and particularly 2200 mg/kg/day. Bodyweight gain was reduced to a toxicologically significant degree in animals of both sexes at 1600 or 2200 mg/kg/day. A slight reduction in food intake was noted for males only at 2200 mg/kg/day. Weight gain for animals previously receiving 2200 mg/kg/day was superior to that of controls in the recovery period such that the intergroup deficit was almost offset within 4 weeks. In Week 5, plasma enzyme activities transaminases were slightly elevated for both sexes at 2200 mg/kg/day plus females at 1600 mg/kg/day. In Week 13 a similar finding was only apparent for males at 2200 mg/kg/day and was associated with periportal hepatocyte hypertrophy seen in the majority of males and an occasional female rat at 2200 mg/kg/day. Following 4 week recovery, the enzyme activity was normal; there was no residual hepatic pathology. An increased incidence and degree of haemosiderosis was noted in the spleen of rats receiving 2200 mg/kg/day at the main kill. After 4 weeks off treatment, the incidence and degree of haemosiderosis was reduced but recovery was not complete. In Week 13, urinary pH levels were reduced for both sexes at 2200 mg/kg/day and for males only at 1600 mg/kg/day following 4 week recovery, the pH levels for males had tended towards recovery but were still reduced for females.

In conclusion, 1000 mg/kg/day was a NOAEL. Dosages of 1600 and 2200 mg/kg/day were tolerated over a 13 week period but induced some or all of changes in blood parameters, minor treatment related pathology and adverse effects on bodyweight gain and food consumption. When selected animals previously receiving 2200 mg/kg/day were maintained off-dose for 4 weeks, all treatment related changes showed a complete or tended towards recovery.

Endpoint conclusion

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

This substance is a reaction mass of dipropylene glycol dibenzoate (DPGDB), diethylene glycol dibenzoate (DEGDB) and triethylene glycol dibenzoate (TEGDB). No testing has been performed on the reaction mass itself but data are available for DPGDB, DEGDB and TEGDB. 

The results from the main studies performed on the three components indicated a “no observed adverse effect level” (NOAEL) of 1000 mg/kg bodyweight/day. Therefore a NOAEL of 1000 mg/kg bodyweight/day is also applicable to the reaction mass.

All main studies on the components were performed according to international test guidelines and in compliance with GLP. Groups of ten rats were dosed by dietary administration with the test substances for a period of 13 weeks at levels 0 (untreated diet control), 250, 1000, 1750, and 2500 mg/kgbw/day for DPGDB and DEGDB and at 0 (untreated diet control), 400, 1000, 1600, and 2200 mg/kgbw/day for TEGDB.  Additional rats were dosed at 0 and the highest dose levels to allow for an assessment of recovery from treatment for four weeks after dosing. Supporting studies were performed on DEGDB only; these were not in compliance with GLP. Studies on dogs (90 days and 4 weeks) and rats (90 days and 3 weeks) were conducted.

 

DPGDB

In the key study no findings of toxicological importance were detected at a dosage of 1000 mg/kg bw/day or below.  Dosages of 1750 or 2500 mg/kgbw/day were tolerated but induced clinical findings which were limited to changes in blood parameters, minor treatment-related pathology and/or adverse effects on bodyweight gain.  When selected animals previously receiving 2500 mg/kgbw/day were maintained off dose for 4 weeks all treatment related changes showed evidence of complete recovery.

DEGDB

In the key study no findings of toxicological importance were detected in this study at a dosage of 1000 mg/kg/day or below.  Dosages of 1750 or 2500 mg/kg/day were tolerated (with one exception - a single mortality at 2500 mg/kg/day) but induced clinical findings changes in blood parameters, minor treatment-related pathology and/or adverse effects on bodyweight gain.  When selected animals previously receiving 2500 mg/kg/day were maintained off dose for 4 weeks all treatment related changes showed evidence of complete recovery.

Two other studies on rats and two studies on dogs (all dietary) support this result:  

- A rat 3 week preliminary study showed no treatment related changes at levels of 0.5 and 1.0% except for lung petechiation in one rat at each level which may have been treatment related. 

- A rat 90 day study showed no treatment related effects between 0.2 – 2.0%. 

- A dog 4 week preliminary study could not define a NOAEL (dose range 0.5 – 5.0%). 

- A dog 90 day study showed no treatment related effects at concentrations between 1000 – 12000 ppm.

 

TEGDB

In the key study, no findings of toxicological importance were detected in this study at a dosage of 1000 mg/kg/day or below.  Dosages of 1600 or 2200 mg/kg/day were tolerated but induced some or all of changes in blood parameters, minor treatment related pathology and adverse effects on bodyweight gain and food consumption. When selected animals previously receiving 2200 mg/kg/day were maintained off-dose for 4 weeks, all treatment related changes showed a complete or tended towards recovery.

Justification for classification or non-classification

On the basis of the findings in the reported subchronic tests (90 -day oral, NOAEL = 1000 mg/kg bw/day), and according to the criteria laid down in EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, the components of the reaction mass are not considered posing danger of serious health damage by prolonged oral exposure a concern for humans and therefore the reaction mass itself is also not classified for repeated dose toxicity.