Propane-1,2-diyl dibenzoate

Endpoint summary

Description of key information

Key short-term and supporting sub-chronic oral repeat dose toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant sub-chronic repeat dose toxicity information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

PGDB

OECD 422 (Rat): NOAEL = 300 mg/Kg bw/day, based on the myofibre degeneration/necrosis observed in the skeletal muscle at 1000 mg/Kg bw/day.

DPGDB

OECD 408 (Rat): NOAEL = 1000 mg/Kg bw/day, based on changes in blood parameters, minor treatment related pathology, and/or adverse effects on bodyweight gain observed in animals dosed at 1750 or 2500 mg/Kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-07-30 to 2014-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

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)

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)

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.

Dose / conc.:

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

Remarks:

Group 1 (Control)
Basis: actual ingested

Dose / conc.:

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

Remarks:

Group 2
Basis: actual ingested

Dose / conc.:

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

Remarks:

Group 3
Basis: actual ingested

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.

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.

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.

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

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

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

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. 

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.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

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.:

0 mg/kg bw/day (nominal)

Remarks:

Control - plain diet

Dose / conc.:

250 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.:

1 750 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.:

2 500 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.
- 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 ofvariance 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

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

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.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

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:

Key data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

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/Kg bw/day. Additional rats were dosed at 0 and 2500 mg/Kg bw/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/Kg bw/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/Kg bw/day day 4 week recovery group.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimish rating 1. Study conducted in a GLP compliant lab according to latest regulatory test Guidelines

System:

other: 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.

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

Key short-term and supporting sub-chronic oral repeat dose toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant sub-chronic repeat dose toxicity information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

In a key OECD Guideline 422 combined repeat dose / reproductive and developmental toxicity screening study (Huntingdon Life Sciences, 2014g; Klimisch score = 1), 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 wereconsistent with dosing injuries. Additionally, some animals at scheduled termination also hadmacroscopic 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 thancontrols 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 formales and high heart weights for females. In the absence of any adverse effects on clinicalcondition 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 increasedwater consumption, low bodyweight gain of males and high motor activity for males andfemales 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) isconsidered 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 necropsyand 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). Afocal, 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. 

In a key OECD Guideline 408 read across 13-week repeated oral dose study (Huntingdon Life Sciences, 1999c; Klimisch score = 1) conducted to determine the effects of prolonged exposure of the test material DPGDB in rats, Crl: (IGS) CD BR rats (5/sex/concentration) were orally administered (via diet) DPGDB at 0 (untreated diet control), 250, 1000, 1750, and 2500 mg/Kg bw/day for a period of 13 weeks. Additional rats were dosed at 0 and 2500 mg/Kg bw/day to allow for an assessment of recovery from treatment 4 weeks after the end of dosing.

 

Clinical observations showed no signs of adverse effects with the exception of a decrease in body weight gain and final body weight 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 bw/day doses. The liver showed minimal to slight liver cell hypertrophy (enlargement) and alterations in associated biochemical parameters. At 1000 mg/Kg bw/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 bw/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 bw/day doses only. Most importantly all treatment related affects were reversible or showed tendency to reverse in the 2500 mg/Kg bw/day day 4 week recovery group.

 

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

In a supporting oral 90-day study (FDRL, 1972; Klimisch score = 2), the test material (Propylene Glycol Dibenzoate) was administered continuously in the diet to FDRL-Wistar derived weanling rats (15/sex/dose) at doses of 0, 100, 500, or 2000 mg/Kg bw/day (equivalent toactual intake of propylene glycol dibenzoate of 126, 633, and 2541 mg/Kg bw/day (combining and averaging box sexes, respectively)) for a period of 90 days. Based on lack of adverse systemic toxicity effects observed in the study, the NOAEL for systemic toxicity was determined to be >2541 mg/Kg bw/day).

Justification for classification or non-classification

Key short-term and supporting sub-chronic oral repeat dose toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant sub-chronic repeat dose toxicity information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB). The justification for read across is presented as an attachment included in Section 13 of the IUCLID dossier.

A STOT RE category classification is based on significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals where the effects are seen in the range 10<C≤100 mg/Kg bw/d. For a 28-day study the guidance values are recommended to be increased by a factor of three such that the range would be 30 < 300 ≤ mg/Kg bw/d. In the OECD 422 study conducted with PGDB, the NOAEL was determiined to be 300 mg/Kg bw/day. Additionally, the NOAEL in the 90-day OECD 408 study conducted with DPGDB was determined to be 1000 mg/Kg bw/day. Taking into consideration the weight of evidence available from the OECD 422 study and the OECD 408 study conducted using structural analogue DPGDB, it is concluded that PGDB does not warrant classification for STOT-RE under the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.