Propane-1,2-diyl dibenzoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-30 to 2014-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations were considered not to have affected the integrity or validity ofthe study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,2-diyl dibenzoate

EC Number:

242-894-7

EC Name:

Propane-1,2-diyl dibenzoate

Cas Number:

19224-26-1

Molecular formula:

C17H16O4

IUPAC Name:

1-(benzoyloxy)propan-2-yl benzoate

Constituent 2

Reference substance name:

1,2-propan-diyl-dibenzoate

IUPAC Name:

1,2-propan-diyl-dibenzoate

Constituent 3

Reference substance name:

K-FLEX* PG

IUPAC Name:

K-FLEX* PG

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Kalama® K-FLEX® PG
- Substance type: Plasticizer
- Physical state: Clear light yellow liquid
- Analytical purity:97.1% propylene glycol esters
- Lot/batch No.: KAKPG43301
- Expiration date of the lot/batch: March 2016
- Storage condition of test material: Ambient temperature under nitrogen

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Performance Materials LLC (USA); Batch No. KAKPG43301
- Expiration date of the lot/batch: March 2016
- Purity test date: 2014-03-05

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, under nitrogen
- Stability under test conditions: The homogeneity and stability offormulations during storage were confirmed as part of another study, Hunt ingdon Life Sciences
study number ORR0088. In that studystabilit y was confirmed at dose levels of 1 and 200 mg/mL for 24 hours in ambient conditions and 15 days when stored refrigerated (nominally +4°C)
- Solubility and stability of the test substance in the solvent/vehicle: Not specified

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid

OTHER SPECIFICS:
Purity: 97.1% - Propylene glycol esters (comprising 88.61% propylene glycol dibenzoate and 8.47% propylene glycol monobenzoate)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: approx.10 weeks
- Weight at study initiation: approx. 333-392g (males), 225-269g (females)
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Number of animals per cage
Pre-pairing: five animals of one sex
Pairing: one male and one female
Males after mating: five males
Gestation: one female
Lactation: one female + litter

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum
- Water (e.g. ad libitum): Potable water fromthe public supply via polycarbonate bottles with sipper tubes ad libitum.
- Acclimation period: at least 5 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light):12 hours light : 12 hours dark.

IN-LIFE DATES: From: 2014-08-04 to 2014-09-08 (Males) or 2014-09-16/29 (Females)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oralgavage route ofadministration was chosen to simulate the condition of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Specimen formulations of PGDB were assessed. Purified water was evaluated at 100 mg/mL and corn oil was assessed at 200 mg/mL. Preparations were physically assessed for colour changes, appearance, haziness or precipitation (as applicable) for up to four hours from preparation. Water produced an off white emulsion which separated quickly. Corn oil made a pale yellow solution.

- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionisation detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 8 μg/mL to 40 μg/mL. Calibration standards and samples each contained an internal standard of biphenyl in acetone at a concentration of ca. 20 μg/mL.

Procedural recovery analysis was performed as a quality control measure and was used to accept or reject analysed batches of samples with reference to the method validation data.

In Weeks 1 and 5 of treatment, freshly prepared test formulations were sampled and submitted for analysis.

Duration of treatment / exposure:

Males: Daily for 2 weeks pre-pairing up to necropsy after minimum of 5 weeks.

Females: Daily for 2 weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.

Frequency of treatment:

F0 females were treated once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration. Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 (Control)
Basis: actual ingested

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2
Basis: actual ingested

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3
Basis: actual ingested

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4
Basis: actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a preliminary toxicity study performed at this laboratory (Huntingdon Life Science study number ORR0088). Administration of PGDB at dose levels up to 1000 mg/kg/daywas well tolerated and any potential treatment related changes were minor and not considered to be adverse at the degree
observed. Therefore in this study dose levels up to the limit dose of 1000 mg/kg/day have been included with low and intermediate dose levels of 100 and 300 mg/kg/day.

- Rationale for animal assignment (if not random): Randomly allocated on arrival.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- During the acclimatisation period, observations of the animals and their cages were recorded at least once per day. During the study animals were visually inspected at least twice daily for evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment started and during each week of treatment onwards - once each week. Females: Gestation phase - Days 0, 6, 13 and 20; Lactation phase – Days 1 and 6. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

BODY WEIGHT: Yes
- Time schedule for examinations: Before treatment commenced (Day 1), weekly thereafter and on the day of necropsy. (Males)
Before treatment commenced (Day 1), weekly before pairing, on Days 0, 6, 13 and 20 after mating, on Days 1, 4 and 7 of lactation and on the day of necropsy. (Females)

FOOD CONSUMPTION : Yes
Time schedule for recording: weekly except during mating (days 15-21). For females weekly until paired for mating, following pairing, on Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 2 prior to pairing
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes
- How many animals: the five lowest numbered surviving males and females per group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 prior to pairing
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes
- How many animals: the five lowest numbered surviving males and females per group
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 5 for the males, between Days 4-6 (before dosing )of lactation for the females
- Dose groups that were examined: the five lowest numbered surviving males in each group and the five lowest numbered surviving lactating females in each group
- Battery of functions tested: sensory activity / grip strength / motor activity

DETAILED PHYSICAL EXAMINATIONS AND ARENA OBSERVATIONS:
- Time schedule for examinations: pre-treatment, then weekly (before dosing on each occasion), on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation (before dosing on each occasion)

Sacrifice and pathology:

GROSS PATHOLOGY: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of necropsy
F0 males: After Week 5 investigat ions completed.
F0 females: Scheduled kill - Day 7 oflactation.
Failing to produce viable litter - Day 25 after mating.
Litters dying before Day 7 - on day last offspring died.
F1 offspring Scheduled kill - Day 7 of age.

Method of kill
F0 animals: Carbon dioxide asphyxiation withsubsequent exsanguinat ion.
Offspring: Intraperitoneal inject ion of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison

HISTOPATHOLOGY: Yes The selected organs were weighed, tissue samples fixed and sections examined microscopically. Pathology procedures for the five lowest numbered surviving males and females per group at scheduled termination. Parameters checked in table [No.4, 5] were examined.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented in the report. All statistical analyses were carried out separately for males and females.

The following data types were analysed at each timepoint separately: Body weight, using absolute weights and gains over appropriate study periods, Food consumption, over appropriate study periods: Blood chemistry and haematology; Organ weights, both absolute and adjusted for terminal body weight; Grip strength and motor activity; Mating performance and fertility; Gestation length and Litter data.

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, clinical pathology, organ weights and litter data:

A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre treatment data, Kruskal-Wallis’ test was used to test for any group differences.

For grip strength, motor activity, clinical pathology and litter data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no dose signs at scheduled observation recording times. The clinical sign of vocalisation was recorded at a higher frequency than usually expected; there was no dose response to this sign.

Additional observations noted included:
1) Increased water consumption for animals receiving 1000 mg/Kg/day.
2) Chin rubbing immediately following dosing. This sign is often associated with a poorly palatable test material and as such is not generally considered to be adverse.
3) Males and females receiving 1000 mg/Kg/day were noted to be ‘grumpy’. This is linked to the vocalisation and aggression recorded during clinical signs and struggling during dose administration. This is a subject ive assessment which is difficult to classify as a formal sign other than if vocalisation or aggression is observed during clinical signs recording.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Group 1 female 73 was found dead on Day14 of gestation. Necropsyfindings included thorax contained clear fluid, pale, gelatinous material adhered to lungs and diaphragm, 2 punctate open areas in the oesophagus. Microscopic examination revealed that this animal had inflammatory change in the thorax involving the thymus, pericardium and the pleura. These changes are consistent with a technical error at dosing resulting in trauma to the tissues.

Group 3 male 26 was killed for welfare reasons on Day 34 of study after showing a decline in clinical condition. Necropsyfindings included soft pale mass invo lving lungs, heart, thymus, oesophagus, aorta and diaphragm, perforated oesophagus (this animal had signs from the 5th September and was monitored closely). Microscopic findings included an abscess in the thorax with inflammatorychanges involving the pleura and pericardium alo ng with haemorrhage in the oesophagus. These changes are consistent with a technical error at dosing resulting in trauma to the tissues.

Group 4 female 43 was killed for welfare reasons on day 1 after mating after showing marked clinical signs. Necropsyfindings included an abnormal oesophagus, thickened and distended, containing pale yellow viscous fluid, with a poorly defined area adjo ining the stomach. The stomach and caecumwere was largely devoid of content. Microscopic examination revealed diffuse inflammatory change in the oesophagus and multifocal alveolitis in the lungs consistent with technical error resulting in mis-dosing of the animal.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of PGDB during the first week of treatment in males.

Females receiving 1000 mg/Kg/day for the same recording period appeared to show increased body weight gain but this difference was not considered to be adverse. Between Days 8-15 before pairing, both males and females receiving 300 or 1000 mg/Kg/day showed statistically significant lower body weight gain compared to the Control, a dose response was not apparent in males. For males receiving 1000 mg/Kg/day, a general trend of lower body weight gain was apparent from Day 8 of the study with the period Days 29-36 (after pairing) attaining statistical significance. As a result overall lower body weight gain in males at the 1000 mg/Kg/day dose level attained statistical significance. No treatment-related effects on body weight or body weight change during gestation or lactation were observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption at any dose level. At 1000 mg/Kg/day, food consumption was generally marginally higher than Control throughout the study for males and before pairing and gestation for females. This type of change is generally not considered to be adverse.

At 1000 mg/Kg/day, food consumption during Days 4-6 of lactation was low.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant changes in haematology parameters assessed for males during week 2 of study were limited to lower eosinophil counts at 1000 mg/Kg/day. Females receiving 1000 mg/Kg/day showed low haematocrit, haemoglobin levels, and red blood cell counts all of which attained statistical significance. Eosinophil counts also were statistically lower than control for females at this dose level.

In the absence of any adverse effects on clinical condition or pathological correlates the above differences were considered not to be adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no consistent effects on blood chemistry parameters between males and females.

Parameters which attained statistical differences to control were; for males receiving 1000 mg/Kg/day high aspartate amino-transferase, high calcium and high phosphorus levels; in females receiving 1000 mg/Kg/day high alanine amino-transferase, low calcium and low albumin levels.

In addition in females receiving 300 or 1000 mg/Kg/day statistically high creatinine and glucose levels were recorded, a dose response was not apparent for either of these parameters and for the glucose measurement the control value was considered to be slightly low as the normal range is 6-10 mmol/L.

In the absence of any adverse effects on clinical condition or pathological correlates the above differences were considered not to be adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no notable changes in behaviour, reflexes or grip strength.

In males and females receiving 1000 mg/Kg/day, motor activity was high, this encompassed both ambulatory(low beam) and rearing (high beam) activity attaining statistical significance for all except low ambulatory scores in females, which is probably because this score is generally higher and more variable during lactation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Body weight adjusted mean liver weights were slightly high for males and females receiving 1000 mg/Kg/day, absolute liver weights were also slightly high in these females.

Statistical changes in adjusted mean organ weights at the 1000 mg/Kg/day dose level affecting only one sex were lower brain weights for males and higher heart weights for females. In the absence of any adverse effects on clinical condition or pathological correlates the above differences were considered not to be adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after 5 weeks of treatment revealed changes in the thorax or thoracic tissues, inflammatory change involving the thymus, pleura, pericardium or oesophagus, indicating dosing trauma in Animals; Group 1 male 31, Group 3 male 22, Group 4 male 2 and Group 4 female 44.

The incidence and distribution ofall the other findings were consistent with the common background findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with PGDB were seen in the liver and skeletal muscle.

Liver: Centrilobular hypertrophy was present in 3/5 males receiving 1000 mg/Kg/day at a minimal level. Increased cytoplasmic rarefaction (glycogen-type vacuolation) was present in 3/5 females at 1000 mg/Kg/day.

Centrilobular hypertrophy is suggestive of an adaptive response to mixed function oxidase induction in the liver (Cattley et al., 2002) and considered to be of limited toxicological significance. The increased cytoplasmic rarefaction (likely due to glycogen deposition) is considered in rodents to be adaption, possibly related to hepatic metabolism of the test substance (Greaves, 2007). These changes correlate with the increase in organ weight noted at necropsy and the change in females maybe linked to the variation in glucose levels in the clinical chemistry findings. Slightly raised blood enzymes seen in the 1000 mg/Kg/day animals may also
be linked to the minor changes in the liver.

Skeletal Muscle: Focal, minimal or multifocal, slight myofibre degeneration/necrosis was present in 3/5 males at 1000 mg/Kg/day. Focal, minimal myofibre degeneration / necrosis was present in 1/5 males at 100 mg/Kg/day and 2/5 males at 300 mg/Kg/day.

This change, present only in males, is seen occasionally as a background change at a minimal level and whilst the significance is not clear it is an unusual finding and a direct relationship to the test substance at a slight, multifocal level in males at 1000 mg/Kg/day cannot be ruled out. This may be linked to the raised aspartate amino-transferase (AST) levels seen in males at 1000 mg/Kg/day.

Incidental findings: There were a number of other findings which are considered to be background changes or related to minor technical errors resulting in oesophageal injury or inflammatorychange in the thorax. These were not considered to be related to PGDB.

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity ofthe various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no dose signs at scheduled observation recording times.
The clinical sign of vocalisation was recorded at a higher frequency than usually expected, there was no dose response to this sign.

Additional observations noted by the technical staff were:
Increased water consumption for the animals receiving 1000 mg/kg/day.
Chin rubbing immediately following dosing. This sign is often associated with a poorly palatable test material and as such is not generally considered to be adverse.
Males and females receiving 1000 mg/kg/day have been noted to be ‘grumpy’. This is linked to the vocalisation and aggression recorded during clinical signs.

MORTALITY
There have been three premature necropsies in this study. 2 animals died of dosing trauma, the third death is due to mis-dosing of the animal.

BODY WEIGHT AND WEIGHT GAIN
Females receiving 1000 mg/kg/day for the same recording period appeared to show an increased bodyweight gain. Between Days 8-15 before pairing of study both males and females receiving 300 or 1000 mg/kg/day statistically significant low bodyweight gain compared to the Control, a dose response was not apparent in males. For males receiving 1000 mg/kg/day a general trend of low bodyweight gain was apparent from Day 8 of study with the period Days 29-36 (after pairing) attaining statistical significance. As a result overall low bodyweight gain in males at the 1000 mg/kg/day dose level attained statistical significance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There is currently no adverse effect on food consumption at any dose level. At 1000 mg/kg/day food consumption was generally higher than Control throughout the study for males and before pairing and gestation for females, this type of change is generally not considered to be adverse. At 1000 mg/kg/day, food consumption during Days 4-6 of lactation was low.

HAEMATOLOGY
There were no statistically significant changes in haematology parameters assessed for males during week 2 of study. Females receiving 1000 mg/kg/day showed low heamatocrit, haemoglobin levels and red blood cell counts all of which attained statistical significance. Eosinophil’s also were statistically lower than Control for females at this dose level.

CLINICAL CHEMISTRY
There were no consistent effects on blood chemistry parameters between males and females.
Parameters which attained statistical differences to Control were; for males receiving 1000 mg/kg/day were high aspartate amino-transferase, high calcium and high phosphorus levels; in females receiving 1000 mg/kg/day high alanine amino-transferase, low calcium and low albumin levels. In addition in females receiving 300 or 1000 mg/kg/day statistically high Creatinine and glucose levels were recorded, a dose response was not apparent for either of these parameters and for the glucose measurement the Control value was considered to be slightly low as the normal range is 6-10 mmol/L.

NEUROBEHAVIOUR
There were no notable changes in behaviour, reflexes or grip strength.
In males and females receiving 1000 mg/kg/day motor activity was high, this encompassed both ambulatory (low beam) and rearing (high beam) activity attaining statistical significance for all except low ambulatory scores in females, which is probably because this score is generally higher and more variable during lactation. This higher activity may be the cause of the generally low weight gain despite the slightly high food consumption in these animals.

ORGAN WEIGHTS
Bodyweight adjusted mean liver weights were slightly high for males and females receiving 1000 mg/kg/day. Statistical changes in adjusted mean organ weights at the 1000 mg/kg/day dose level affecting only one sex were lower brain weights for males and higher heart weights for females.

GROSS PATHOLOGY
At scheduled necropsy three males and one female had post mortem findings of concern. These findings show no relationship to treatment but we would not normally expect to see these changes at scheduled necropsy. These findings are considered to suggest dosing trauma.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects: histopathology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

musculoskeletal system

Organ:

myofibres

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 6. Body weight - group mean values (g) for males (F0)
Group (mg/Kg/day)		Day 1	Day 8	Day 15	Day 22	Day 29	Day 36	Change 1-8	Change 8-15	Change 15-22	Change 22-29	Change 29-36	Change 1-15	Change 1-36
Statistical Test		Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
0 (Control)	Mean	363	405	447	466	498	518	42	41	20	32	20	84	155
	SD	9.4	14.7	23.3	30.1	35.0	43.2	8.5	10.1	8.6	7.4	12.0	17.6	36.6
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
100	Mean	356	398	433	451	482	504	42	35	18	31	22	77	147
	SD	11.9	19.3	25.7	29.5	33.3	35.8	9.8	9.9	8.4	8.1	4.7	17.3	27.8
	N	10	10	10	10	10	10	10	10	10	10	10	10	10
300	Mean	356	396	426	448	473	502	40	30*	22	25	20	70	144
	SD	8.5	14.8	18.7	22.0	34.8	28.7	7.8	8.4	11.9	19.9	8.8	13.1	26.1
	N	10	10	10	10	10	9	10	10	10	10	9	10	9
1000	Mean	363	402	434	452	479	487	39	32*	17	28	8**	71	124*
	SD	17.5	23.1	26.6	22.3	27.7	28.4	7.9	7.6	10.2	13.3	6.4	10.5	23.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10

Av - Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 7. Body weight - group mean values (g) for females before pairing (F0)
Group (mg/Kg/day)		Day 1	Day 8	Day 15	Change 1-8	Change 8-15	Change 1-15
Statistical Test		Av	Wi	Wi	Wi	Wi	Wi
0 (Control)	Mean	243	248	259	5	11	16
	SD	9.7	10.7	10.5	7.9	5.8	9.4
	N	10	10	10	10	10	10
100	Mean	245	251	258	5	7	12
	SD	14.3	14.5	15.2	3.6	6.4	6.2
	N	10	10	10	10	10	10
300	Mean	239	246	250	7	4*	11
	SD	6.4	8.3	12.0	7.4	5.7	10.5
	N	10	10	10	10	10	10
1000	Mean	237	250	256	13**	6*	19
	SD	7.1	7.9	8.1	6.2	6.3	6.7
	N	10	10	10	10	10	10

Av - Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 8. Food consumption - group mean values (g/animal/day) for females during gestation (F0)
Group (mg/Kg/day)		Day 0 - 5	Day 6 - 12	Day 13 - 19
Statistical Test		Wi	Wi	Wi
0 (Control)	Mean	19	22	24
	SD	1.6	1.5	3.0
	N	10	10	9
100	Mean	20	22	24
	SD	2.6	3.6	2.6
	N	10	10	10
300	Mean	21	22	23
	SD	3.1	2.4	2.3
	N	10	10	10
1000	Mean	22*	24	25
	SD	2.3	2.8	2.3
	N	8	8	8

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

Table 9. Food consumption - group mean values (g/animal/day) for females during lactation (F0)
Group (mg/Kg/day)		Day 1 – 3	Day 4 - 6
Statistical Test		Wi	Wi
0 (Control)	Mean	34	50
	SD	7.3	5.9
	N	9	9
100	Mean	35	48
	SD	6.7	5.5
	N	10	10
300	Mean	37	51
	SD	5.5	4.2
	N	10	10
1000	Mean	31	41**
	SD	5.4	5.1
	N	6	6

Wi - Treated groups compared with Control using Williams’ test.

**p<0.01

Table 10. Haematology - group mean values during Week 2 before pairing (F0)
Group (mg/Kg/day)		Eosinophils (E) X 10^9/L	Haematocrit (Hct) (L/L)	Haemoglobin (Hb) (g/dL)	Erythrocyte Count (RBC) X 10^12/L
Statistical Test		Wi	Wi	Wi	Wi
Males
0 (Control)	Mean	0.17	0.465	15.0	7.73
	SD	0.0068	0.0081	0.27	0.350
	N	5	5	5	5
100	Mean	0.15	0.459	14.9	7.60
	SD	0.043	0.0090	0.31	0.281
	N	5	5	5	5
300	Mean	0.14	0.461	14.9	7.42
	SD	0.050	0.0129	0.48	0.246
	N	5	5	5	5
1000	Mean	0.08**	0.463	15.0	7.88
	SD	0.0020	0.0108	0.46	0.414
	N	5	5	5	5
Females
0 (Control)	Mean	0.18	0.460	15.3	8.03
	SD	0.077	0.0076	0.31	0.196
	N	5	5	5	5
100	Mean	0.09	0.445	14.8	7.74
	SD	0.027	0.0074	0.24	0.319
	N	5	5	5	5
300	Mean	0.16	0.451	14.9	7.71
	SD	0.073	0.0175	0.71	0.262
	N	5	5	5	5
1000	Mean	0.09*	0.427**	14.2**	7.35**
	SD	0.025	0.0154	0.66	0.274
	N	5	5	5	5

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 11. Blood chemistry - group mean values during Week 2 before pairing (F0)
Group (mg/Kg/day)		AST (U/L)	ALT (U/L)	Na (mmol/L)	Ca (mmol/L)	Inorganic Phosphorous (mmol/L)	Creatinine (µmol/L)	Glucose (mmol/L)	Albumin (g/L)
Statistical Test		Wi		Du	Wi	Wi	Wi	Wi	Wi
Males
0 (Control)	Mean	70	61	141	2.69	2.45	28	7.17	35
	SD	5.9	10.8	0.8	0.054	0.140	1.8	0.394	1.2
	N	5	5	5	5	5	5	5	5
100	Mean	75	49	143**	2.70	2.42	29	6.80	25
	SD	2.4	8.1	0.8	0.081	0.172	3.3	1.206	1.4
	N	5	5	5	5	5	5	5	5
300	Mean	65	55	141	2.78	2.59	29	7.47	34
	SD	4.1	9.6	0.9	0.081	0.081	4.1	1.308	0.9
	N	5	5	5	5	5	5	5	5
1000	Mean	91**	76	142	2.81*	2.96**	29	7.37	34
	SD	12.5	18.9	1.1	0.061	0.212	2.9	0.868	0.9
	N	5	5	5	5	5	5	5	5
Females
0 (Control)	Mean	71	50	142	2.73	2.13	31	5.72	38
	SD	4.4	5.2	1.1	0.050	0.139	1.3	0.689	1.6
	N	5	5	5	5	5	5	5	5
100	Mean	67	54	142	2.71	1.89	33	7.13	37
	SD	7.6	8.9	0.7	0.102	0.145	2.4	0.891	1.7
	N	5	5	5	5	5	5	5	5
300	Mean	80	49	141	2.75	2.13	37*	8.82**	37
	SD	17.1	11.5	0.7	0.043	0.135	4.6	1.818	1.5
	N	5	5	5	5	5	5	5	5
1000	Mean	81	69*	141	2.57**	2.23	35*	8.28**	35*
	SD	9.7	19.5	1.7	0.108	0.181	3.6	0.560	1.9
	N	5	5	5	5	5	5	5	5

Du - Treated groups compared with Control using Dunnett’s test.

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 12. Motor activity - group mean scores (beam breaks) during Week 5 of treatment (F0) - Males
Group (mg/Kg/day)	No. of animals	Beam Level	Time (Minutes)
			6	12	18	24	30	36	42	48	54	60	Total
Statistical Test			Wi	Wi	Wi	Wi	Sh	Fe	Fe	Fe	Wi	Wi	Wi
0 (Control)	5	High	95.8	65.6	3.6	10.6	0.8	0.0	0.8	5.0	7.2	11.2	200.6
		SD	43.0	26.6	3.6	16.6	1.8	0.0	1.8	11.2	10.0	10.8	60.3
100	5	High	115.4	83.4	22.0	14.8	27.0	4.6	4.4	5.4	39.4	59.0	375.4
		SD	65.2	24.8	17.4	18.1	32.0	6.4	9.8	11.5	45.9	51.4	206.2
300	5	High	101.0	60.8	42.6**	15.6	6.2	0.0	0.0	0.6	14.6	7.0	248.2
		SD	19.3	38.2	25.5	19.8	8.5	0.0	0.0	1.3	31.5	15.7	62.1
1000	5	High	122.4	73.4	68.0**	52.4**	24.2*	20.6	17.2	6.4	47.6	41.6	473.8**
		SD	23.4	14.8	24.3	17.3	20.8	38.4	35.2	14.3	26.0	53.9	155.7
Statistical Test			Wi	Wi	Wi	Wi	Wi	Sh	Wi	Wi	Wi	Wi	Wi
0 (Control)	5	Low	232.2	165.8	55.2	42.6	8.4	0.4	11.4	19.0	39.2	55.6	629.8
		SD	87.2	36.9	44.7	36.3	17.7	0.9	16.3	41.4	52.8	56.3	214.1
100	5	Low	210.2	162.8	77.0	70.2	50.8	30.6	21.0	36.0	60.2	84.2	803.0
		SD	59.0	39.5	61.2	64.6	50.4	42.6	46.4	35.6	23.8	54.1	305.2
300	5	Low	233.2	162.2	119.2	71.2	28.0	4.4	40.6	11.6	49.8	29.6	749.8
		SD	42.2	85.5	46.8	57.7	31.9	4.4	78.0	17.2	99.2	58.1	238.5
1000	5	Low	259.8	193.8	170.4**	137.4**	109.6**	51.2*	44.4	23.8	97.8	79.4	1167.6**
		SD	46.8	47.8	39.9	34.3	48.1	32.0	26.8	35.1	34.3	39.7	40.6

Fe - Treated groups compared with Control using Fisher’s Exact test.

Sh - Treated groups compared with Control using Shirley’s test.

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 13. Motor activity - group mean scores (beam breaks) at Day 4-6 of lactation (F0) - Females
Group (mg/Kg/day)	No. of animals	Beam Level	Time (Minutes)
			6	12	18	24	30	36	42	48	54	60	Total
Statistical Test			Wi	Sh	Sh	Wi	Wi	Wi	Sh	Wi	Sh	Wi	1Wi
0 (Control)	5	High	67.2	14.4	9.6	12.8	9.0	12.8	8.0	7.2	10.2	4.6	155.8
		SD	12.3	11.8	10.4	15.2	12.4	20.5	11.3	9.9	14.9	10.3	88.4
100	5	High	73.6	16.6	6.8	9.2	8.2	17.6	50.2	21.6	10.4	15.0	229.2
		SD	19.0	6.2	7.4	6.8	17.2	14.6	62.5	39.5	23.3	33.5	155.6
300	5	High	79.8	26.0	5.2	10.2	20.4	28.2	23.4	10.2	3.6	19.4	226.4
		SD	27.2	25.2	7.0	14.6	21.7	21.7	13.2	9.9	4.9	20.6	89.7
1000	5	High	131.0**	47.8	35.0	30.0	49.4	34.8	56.4*	41.2	31.6	34.4	491.6*
		SD	35.4	44.5	37.4	31.9	55.6	35.8	59.4	37.0	59.1	44.1	401.4
Statistical Test			Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
0 (Control)	5	Low	216.0	102.8	80.6	84.2	67.0	72.6	44.0	32.2	41.0	41.0	781.4
		SD	32.0	37.1	42.8	47.3	74.1	75.5	49.9	49.9	39.0	54.2	159.6
100	5	Low	187.8	91.8	83.4	80.8	44.2	114.2	58.6	48.0	11.0	35.0	754.9
		SD	61.2	34.8	48.9	42.5	41.3	25.4	31.0	31.0	16.1	46.3	159.6
300	5	Low	170.4	99.2	63.6	52.6	89.2	81.2	84.8	46.4	23.6	62.8	773.8
		SD	74.6	60.6	37.1	34.3	68.3	33.8	24.7	30.8	21.1	63.3	279.9
1000	5	Low	218.2	118.4	119.4	60.6	82.4	86.2	79.4	90.8*	71.2	60.0	986.6
		SD	42.3	48.3	33.7	55.2	59.7	36.8	35.2	28.6	39.8	44.1	327.8

1 - Data were log transformed for the statistical analysis

Sh - Treated groups compared with Control using Shirley’s test.

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

**p<0.01

Table 14. Organ weights - group mean absolute and adjusted values (g) for Males (F0) and for Females on Day 7 of lactation (F0)
Group (mg/Kg/day)
Unadjusted Means (Males)
Statistical Test	Wi	Terminal Body Weight	Brain	Liver	Heart
0 (Control)	Mean	517	2.14	21.52	1.554
	SD	44	0.08	3.36	0.088
	N	10	6	6	6
100	Mean	505	2.16	23.69	1.752
	SD	37	0.11	4.27	0.157
	N	10	5	5	5
300	Mean	503	2.16	21.58	1.600
	SD	31	0.09	1.96	0.074
	N	9	5	5	5
1000	Mean	489	2.00	22.36	1.564
	SD	28	0.11	2.41	0.141
	N	10	6	6	6
Adjusted Means (Males)
Statistical Test			Wi	Wi	Wi
0 (Control)	Mean		2.14	21.34	1.551
100	Mean		2.16	22.98	1.739
300	Mean		2.16	21.52	1.599
1000	Mean		2.00*	23.18*	1.579
Unadjusted Means (Females)
Statistical Test	Wi	Terminal Body Weight	Brain	Liver	Heart
0 (Control)	Mean	349	1.95	17.33	1.181
	SD	23	0.03	1.07	0.066
	N	9	5	5	5
100	Mean	348	1.97	17.66	1.062
	SD	21	0.05	1.79	0.112
	N	10	5	5	5
300	Mean	347	2.01	17.28	1.166
	SD	15	0.08	0.72	0.109
	N	10	5	5	5
1000	Mean	333	1.93	19.14	1.235
	SD	19	0.10	2.56	0.133
	N	7	5	5	5
Adjusted Means (Females)
Statistical Test			Wi	Wi	Wi
0 (Control)	Mean		1.94	17.12	1.163
100	Mean		1.96	17.57	1.054
300	Mean		2.00	17.08	1.148
1000	Mean		1.94	19.65*	1.280*

Wi - Treated groups compared with Control using Williams’ test.

*p<0.05

Table 15. Histopathology - group distribution of findings (F0)
Tissue and Finding	Sex	Males				Females
	Group (mg/Kg/day)	0 (Control)	100	300	1000	0 (Control)	100	300	1000
	Number	10	10	9	10	9	10	10	7
Liver	No. Examined	5	5	5	5	5	5	5	5
Bile Duct Hyperplasia	Minimal	0	0	0	0	1	0	0	0
	Total	0	0	0	0	1	0	0	0
Hepatocyte Hypertrophy, Centrilobular	Minimal	0	0	0	3	0	0	0	0
	Total	0	0	0	3	0	0	0	0
Hepatocyte Vacuolation, Centrilobular	Minimal	1	0	1	0	0	0	0	0
	Slight	2	5	1	0	0	0	0	0
	Total	3	5	2	0	0	0	0	0
Increased Cytoplasmic Rarefaction	Slight	0	0	0	0	0	0	0	3
	Total	0	0	0	0	0	0	0	3
Inflammation, Focal	Minimal	4	5	5	3	5	4	5	2
	Slight	1	0	0	0	0	1	0	0
	Total	5	5	5	3	5	5	5	2
Skeletal Muscle	No. Examined	5	5	5	5	5	0	0	5
	Minimal	0	1	2	1	0	0	0	0
	Slight	0	0	0	2	0	0	0	0
	Total	0	1	2	3	0	0	0	0

Applicant's summary and conclusion

Conclusions:

Based on the results observed, the no observed adverse effect level (NOAEL) for systemic toxicity was determined to be 300 mg/Kg bw/day, based on the myofibre degeneration / necrosis observed in the skeletal muscle at 1000 mg/Kg bw/day. 

Executive summary:

In a key OECD Guideline 422 combined repeat dose / reproductive and developmental toxicity screening study, the systemic toxicity potential of the test material (PGDB) was assessed in Crl:CD(SD) rats (10/sex/dose) following oral gavage administration at doses of 0, 100, 300 or 1000 mg/Kg bw/day over a period of at least five weeks. Male rats were treated daily two weeks before pairing up to necropsy after a minimum of five consecutive weeks and female rats were treated daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted control group received the vehicle, corn oil.

 

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight, macroscopic and microscopic investigations were undertaken for all adult animals.

 

There were 3 premature decedents, all with changes in the thorax or thoracic tissues, which were consistent with dosing injuries. Additionally, some animals at scheduled termination also had macroscopic and microscopic evidence of similar changes indicating dosing trauma. There was no indication of a dose related trend in these changes and the test material was not considered to be the cause of these difficulties. The use of non-standard dosing equipment, due to incompatibilities of the test material with rubber catheters was attributed to be the cause.

 

Treatment with the test material at dose levels up to 1000 mg/Kg bw/day was well tolerated. No adverse treatment-related effects were observed on clinical condition , dosing observations, bodyweight performance of females, food consumption, behaviour, and reflexes or grip strength. Gross necropsy did not reveal any remarkable findings. There was no effect of treatment on oestrus cycle, mating ability of animals, fertility, sex ratio or offspring clinical signs.

 

Haematology assessment revealed low haematocrit, haemoglobin levels, and red blood cell counts for females receiving 1000 mg/Kg bw/day. Eosinophil counts were also observed to be lower than controls for males and females at this dose level. Males receiving 1000 mg/Kg bw/day exhibited high calcium and phosphorus levels and females receiving 1000 mg/Kg bw/day exhibited low calcium and albumin levels. Additionally, females receiving 300 or 1000 mg/Kg bw/day exhibited high creatinine levels although a dose response was not apparent.

 

Adjusted mean organ weights at the 1000 mg/Kg bw/day dose level included low brain weights for males and high heart weights for females. In the absence of any adverse effects on clinical condition or pathological correlates, the above differences were considered not to be adverse. Other effects attributed to the test material at the 1000 mg/Kg bw/day level included increased water consumption, low bodyweight gain of males and high motor activity for males and females including both, ambulatory(low beam) and rearing (high beam) activity.

 

At terminal sacrifice, changes were observed in the liver of the 1000 mg/Kg bw/day animals. Centrilobular hypertrophy was observed in males and increased cytoplasmic rarefaction was observed in females. Centrilobular hypertrophy is suggestive of an adaptive response to mixed function oxidase induction in the liver (Cattley et al., 2002) and considered to be of limited toxicological significance. The increased cytoplasmic rarefaction (likely due to glycogen deposition) is considered in rodents to be adaption, possibly related to hepatic metabolism of the test substance (Greaves, 2007). These changes correlate with the increase in organ weight noted at necropsy and the change in females maybe linked to the variation in glucose levels in the clinical chemistry findings. Slightly raised blood enzymes seen in the 1000 mg/Kg bw/day animals may also be linked to the minor changes in the liver.

 

In males dosed at 1000 mg/Kg bw/day, at terminal sacrifice, myofibre degeneration / necrosis in the skeletal muscle was observed in 3/5 animals (focal, minimal in one and multifocal, slight in two). A focal, minimal change was present in 1/5 animals at 100 mg/Kg bw/day and 2/5 animals at 300 mg/Kg bw/day. This change, present only in males, is seen occasionally as a background change at a minimal level and whilst the significance is not clear, it was an unusual finding and a direct relationship to the test substance at a slight, multifocal level in males at 1000 mg/Kg bw/day could not be ruled out. This may be linked to the raised aspartate amino-transferase (AST) levels seen in males at 1000 mg/Kg bw/day.

 

Based on the results observed, the no observed adverse effect level (NOAEL) for systemic toxicity was determined to be 300 mg/Kg bw/day, based on the myofibre degeneration / necrosis observed in the skeletal muscle at 1000 mg/Kg bw/day.