Propane-1,2-diyl dibenzoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

PGDB

OECD 422 (Rat): Reproductive toxicity NOAEL = 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

DPGDB

OECD 416 (Rat): Reproductive toxicity NOEL = 10000 ppm (equivalent to a minimum estimated daily achieved dosage of 500 mg/Kg bw/d).

Benzoic Acid

OECD 443 (Rat):Reproductive toxicity NOAEL = 15000 ppm

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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. 333-392 g (males), 225-269 g (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

Vehicle:

corn oil

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged singly and males were housed 5 per cage: male/female separation on the day when mating evidence detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

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.

Duration of treatment / exposure:

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

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

Frequency of treatment:

Daily - 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.

Details on study schedule:

- Age at mating of the mated animals in the study: approx. 12 weeks

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.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Visually inspected at least twice daily for evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment for all adults and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for adult Females.

BODY WEIGHT: Yes
- Time schedule for examinations: before dosing on the Day that treatment commences and weekly thereafter. On day of necropsy. Females: Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION : Yes
Time schedule for recording: weekly except during mating (Days 15 to 21) for Males. For females after mating food consumption schedule will match body weight schedule, Days 0-5, 6-12, 13-19 after mating; 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: Yes 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: Yes 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 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)

Oestrous cyclicity (parental animals):

Dry smears: Daily for 15 days before pairing, using cotton swabs moistened with saline.
Wet smears After pairing until evidence of mating was observed, using pipette lavage.

Sperm parameters (parental animals):

Parameters examined in all P male parental generations:
For the assessment of the testes, a detailed qualitative examinat ion was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

PARTURITION OBSERVATIONS AND GESTATION LENGTH
Duration of gestation: Time elapsing between mating and commencement of parturition.
Parturition observations: From Day 20 after mating, animals checked 3 times daily for evidence of parturition. If difficulties observed, progress of parturition process monitored. Numbers of live and dead offspring recorded.

PARAMETERS EXAMINED DURING LITTERING
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
Clinical observations: 24 hours after birth and then daily for evidence of ill-health or reaction to maternal treatment.
Litter size: Daily on Days 1-7 of age.
Sex ratio: Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Offspring at scheduled termination (Day 7 ofage): Examined externally; those offspring deemed normal were discarded without further macroscopic examination. Any externally abnormal offspring were examined internally and any abnormal tissues were retained.

Decedents: Where possible fresh macroscopic examination (external and internal) with assessment of stomach for milk contents. Abnormal tissues retained.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after Week 5 investigations completed (3 weeks after beginning of pairing)
- F0 Females failing to mate: Day 25 after last day of pairing
- F0 Females failing to produce viable litter: Day 25 after mating
- F0 Females whose litters died before Day 7: On day last offspring dies.
All other F0 Females sacrificed on Day 7 of lactation

GROSS NECROPSY
- Detailed macroscopic examination and premature adult decedents
- Males and females: Detailed macroscopic examination will be performed for evidence of adverse reaction to treatment. For females, the number of uterine implantation sites were recorded.
- Female whose litter dies before Day 7 of lactation: Mammary tissue appearance
- Offspring: Examined externally, any externally abnormal offspring were also examined internally. Abnormal tissues retained.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs weighed and tissues retained as specified in Table 4 and Table 5.
Processing – Full List: All animals killed or dying prematurely. The five lowest numbered surviving males and females for animals in each
Group at the scheduled termination.
Processing – All animals.

Postmortem examinations (offspring):

SACRIFICE
-All F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- for offspring, where possible, fresh macroscopic examination (external and internal) with an assessment of stomach for milk content.

HISTOPATHOLOGY
- Examined externally, if found to be normal offspring discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues retained.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the report tables. For some parameters, including oestrous cycles, the similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
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 (Bartlett 1937) 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 (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other analyses the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.

Reproductive indices:

Pre-coital interval calculated for each female as time between first pairing and evidence of mating. The mating performance, conception rate, fertility index and gestation length was calculated for all animals (as appropriate).

Offspring viability indices:

The litter size, the post-implantation survival index, live birth index and viability index were noted or calculated. The sex ratio of each litter was also calculated

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.

Dermal irritation (if dermal study):

not examined

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 involving 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 inflammatory changes 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 caecum were 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

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

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment on oestrus cycles, the ability ofthe animals to mate or fertility.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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).

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of treatment on oestrus cycles, the ability ofthe animals to mate or fertility.

In the Control, 100 and 300 mg/Kg/day groups all pairs mated within 4 days of pairing. However, at 1000 mg/Kg/day one pair mated within 5-8 days and another pair mated within 13-14 days. In addition, at 1000 or 300 mg/Kg/day, there were fewer females with solid masses of sperm (category 4) in the vaginal smear at mating compared with Controls and the 100 mg/Kg/day group, and more females with few/occasional/no sperm (Categories 1-3) in the vaginal smear; the differences attained statistical significance at 1000 mg/Kg/day.

There was no effect of treatment at 100 or 300 mg/Kg/dayon the length of gestation. However, at 1000 mg/Kg/day 3/8 females had a 23.5 day length of gestation compared with 1/29 females in the Control, 100 or 300 mg/Kg/day groups, this shift was within the normal range of 22-23.5 days. There was no effect of treatment on the gestation index.

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.

HISTOPATHOLOGY
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.

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: Systemic 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

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed that could be attributed to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

There was a slight decrease in live birth index survival, mainly due to the total litter loss in one female (no. 46) in the 1000 mg/Kg/day dose group on Day 1 of age. However, even when the this litter is excluded the number of offspring dying between birth and Day7 ofage were 4, 5, 2 and 10 for Control, 100, 300 and 1000 mg/Kg/day groups respectively. The viability index was similar to Control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female offspring body weight on Day1 of age was low at the 1000 mg/Kg/day. Body weight gain thereafter was slightly low at 300 or 1000 mg/Kg/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Among decedent offspring, there were no findings considered to be related to treatment. A total of eight decedent off spring from two litters in the 1000 mg/kg/day were found to have no milk in stomach but this is a common finding in decedent offspring and was seen in one Control pup.

At scheduled termination on Day 7 of age, all off spring in one litter in the 1000 mg/kg/day group were found to have thin build and no milk in stomach while a pup in another litter was found to be smaller than its siblings.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex Ratio: There was no effect on sex ratio.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING) No effect. The viability index was similar to Control.

CLINICAL SIGNS (OFFSPRING) There were no clinical signs observed that could be attributed to treatment.

BODY WEIGHT (OFFSPRING) Male and female offspring body weight on Day 1 of age was low at the 1000 mg/kg/day. Body weight gain thereafter was slightly low at 300 or 1000 mg/kg/day.

SEXUAL MATURATION (OFFSPRING) Not examined

ORGAN WEIGHTS (OFFSPRING) Not examined

GROSS PATHOLOGY (OFFSPRING) Among decedent offspring, there were no findings considered to be related to treatment. A total of eight decedent offspring from two litters in the 1000 mg/kg/day were found to have no milk in stomach but this is a common finding in decedent offspring and was seen in one Control pup.
At scheduled termination on Day 7 of age, all offspring in one litter in the 1000 mg/kg/day group were found to have thin build and no milk in stomach while a pup in another litter was found to be smaller than its siblings.

HISTOPATHOLOGY (OFFSPRING) Not examined

OTHER FINDINGS (OFFSPRING) There was a slight decrease in live birth index and a small increase in the number of offspring dying between birth and Day 7 of age for the group receiving 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects low off spring growth and post-natal offspring mortality

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

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 pairwiset-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 pairwiset-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

Table 16. Mating performance and fertility - group values (F0)
Group (mg/Kg/day)	Number Paired	Number Mating	Number Achieving Pregnancy	Percentage Mating	Conception Rate (%)	Fertility Index (%)
Males
Statistical Test:					Ca	Ca
0 (Control)	10	10	10	100	100	100
100	10	10	10	100	100	100
300	10	10	10	100	100	100
1000	9#	9#	8	100	89	89
Females
Statistical Test:					Ca	Ca
0 (Control)	10	10	10	100	100	100
100	10	10	10	100	100	100
300	10	10	10	100	100	100
1000	9#	9#	8	100	89	89

# Excludes one male and one female - female found dead one day after mating, unable to assess pregnancy status

Ca Treated groups compared to Control using Cochran-Armitage test

Table 17. Sperm count estimates from vaginal smears at mating - group values (F0)
Group (mg/Kg/day)	Number of Animals		Sperm Count Category ῶ
Statistical test: Lt			0	1	2	3	4
0	10	N	0	0	1	1	8
		(N%)			(10)	(10)	(80)
100	10	N	1	0	0	1	8
		(N%)	(10)			(10)	(80)
300	10	N	3	0	0	2	5
		(N%)	(30)			(20)	(50)
1000*	10	N	1	2	1	2	4
		(N%)	(10)	(20)	(10)	(20)	(40)

ῶ        0 No sperm

1 Occasional sperm

2 Continuous few sperm

3 Many scattered sperm

4 Solid masses of sperm

 

Table 18. Litter size - group mean values (F1)
Dose Group (mg/Kg/day)		Implantations	Total Litter Size	Live Litter Size on Day of Age
			Day 1†	1	4	7
Statistical Test:		Wi	Wi	Wi	Wi	Wi
0 (Control)	Mean	15.4	13.7	13.3	13.2	13.2
	SD	1.4	2.7	3.0	3.2	3.2
	N	9	9	9	9	9
100	Mean	16.0	14.8	14.4	14.3	14.3
	SD	2.0	2.5	3.0	2.8	2.8
	N	10	10	10	10	10
300	Mean	16.5	15.7	15.5	15.5	15.5
	SD	1.4	1.3	1.2	1.2	1.2
	N	10	10	10	10	10
1000	Mean	14.8	14.0	13.1	12.6	12.6
	SD	2.0	2.7	3.1	3.6	3.6
	N	8	8	7	7	7

† May include offspring that died prior to the designated Day 1 of age. Unsexed offspring missing prior to Day 1 are not accounted for.

Table 19. Offspring survival indices - group mean values (F1)
Dose Group (mg/Kg/day)		Post Implantation Survival Index (%)	Live Birth Index (%)	Viability Index (%)
Statistical Test:		Wi	Sh	Fe
0 (Control)	Mean	87.7	97.0	98.6
	N	9	9	9
100	Mean	92.4	96.8	99.4
	N	10	10	10
300	Mean	95.4	98.8	100.0
	N	10	10	10
1000	Mean	94.4	81.5	95.1
	N	8	8	7

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.

Table 20. Body weight and bodyweight change - group mean values (g) for male offspring (F1)
Dose Group (mg/Kg/day)		Day of Age			Change
		1	4	7	1-4	4-7	1-7
Statistical Test:		Wi	Wi	Wi	Wi	Wi	Wi
0 (Control)	Mean	7.2	10.3	14.7	3.1	4.5	7.5
	SD	0.9	1.4	1.9	0.7	0.6	1.2
	N	9	9	9	9	9	9
100	Mean	7.2	10.1	14.4	2.9	4.3	7.2
	SD	0.6	1.2	2.0	0.8	0.9	1.6
	N	10	10	10	10	10	10
300	Mean	7.0	9.5	13.4	2.5	3.9	6.4
	SD	0.4	0.7	1.4	0.5	0.6	1.1
	N	10	10	10	10	10	10
1000	Mean	6.4*	8.9*	12.5*	2.5	3.6*	6.1*
	SD	0.6	0.7	0.6	0.8	0.8	1.1
	N	6	6	6	6	6	6

*p<0.05

 

Table 21. Body weight and bodyweight change - group mean values (g) for female offspring (F1)
Dose Group (mg/Kg/day)		Day of Age			Change
		1	4	7	1-4	4-7	1-7
Statistical Test:		Wi	Wi	Wi	Wi	Wi	Wi
0 (Control)	Mean	6.7	9.8	14.3	3.1	4.5	7.6
	SD	0.7	1.2	1.7	0.6	0.6	1.1
	N	9	9	9	9	9	9
100	Mean	6.7	9.4	13.6	2.7	4.2	7.0
	SD	0.5	1.0	1.7	0.7	0.8	1.5
	N	10	10	10	10	10	10
300	Mean	6.7	9.1	12.8*	2.4	3.7*	6.1*
	SD	0.5	0.8	1.1	0.5	0.4	0.8
	N	10	10	10	10	10	10
1000	Mean	6.1	8.5*	12.1**	2.4	3.5**	6.0*
	SD	0.5	0.6	0.6	0.6	0.7	0.9
	N	6	6	6	6	6	6

*p<0.05

**p<0.01

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. The NOAEL for reproductive and developmental toxicity was also determined to be 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

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.

 

Reproductive endpoints which were affected by oral gavage administration of PGDB included; an extended pre-coital interval for two animals and slightly lower mating evidence (sperm evidence in vaginal smears) for animals receiving 1000 mg/Kg bw/day. There was a slight decrease in live birth index and a small increase in the number of offspring dying between birth and Day 7 of age for the group receiving 1000 mg/Kg bw/day.

 

Male and female offspring body weight on Day 1 of age was low at 1000 mg/Kg bw/day; this was despite a tendency towards a longer gestation length which was probably secondary to restricted intra uterine growth. Offspring body weight gain thereafter remained slightly low at 300 or 1000 mg/Kg bw/day. Macroscopic examination at scheduled termination on Day 7 of age confirmed this with, a number of offspring in the 1000 mg/Kg bw/day group noted to be of thin build and with no milk in stomach. As the effects on offspring survival were limited to the 1000 mg/Kg bw/day group, the effects at 300 mg/Kg bw/day were considered not to be adverse at the degree observed.

 

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. The NOAEL for reproductive and developmental toxicity was also determined to be 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 1999 - 13 January 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The substance has identity with Benzoflex 9-88. It is clear colourless liquid and can be stored at ambient temprature.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, England.
- Age at study initiation: (P) x 6 wks; (F1) x 6 wks
- Weight at study initiation: (P) Males 208 ± 18.7, 210 ± 19.6, 209 ± 22.7 and 209 ± 24.1 g (Groups 1 to 4 respectively) and for the females 163 ± 16.8, 162 ± 14.7, 162 ± 16.0 and 163 ± 14.3 g (Groups 1 to 4 respectively)
- Housing: Stainless steel or HDP bodies with lids of stainless steel grid.
- Diet: Commercially available laboratory animal diet LAD 2 SQC from Special Diet services Limited, Witham, Essex, England, ad libitum
- Water: ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Air changes (per hr): The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to the atmosphere and not recirculated.
- Photoperiod : 12 hrs dark / 12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets containing the test material were freshly prepared at regular intervals during the study in batches covering up to two weeks of treatment and prepared up to one week in advance of the first day of feeding.
- Mixing appropriate amounts with (Type of food): Commercially available powdered laboratory animal diet, LAD 2 SQC.
- Storage temperature of food: The homogeneity and the stability, during ambient storage for 22 days, were confirmed for DPGDB in LAD 2 at nominal concentrations of 500 ppm and 25000 ppm. The storage period represented the maximum time from preparation to completion of use.

Quality control of dosage form: Information on the homogeneity of mixing stability and concentration of the test material in the diet was determined by Huntingdon Life Sciences The homogeneity and the stability during ambient temperature storage for 22 days were confirmed for DPGDB in LAD 2 formulation at nominal concentrations of 500 ppm and 25000 ppm (Huntingdon Life Sciences Report VCL315/990088).
The storage period represented the maximum time from preparation to completion of use.

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: Up to 3 weeks
- Proof of pregnancy: Each morning following pairing the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage ofthe oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Stainless steel or HDP bodies with lids of stainless steel grid.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (nominally 200 g) of treated diets were taken at approximately 10-week intervals approxiamtely equivalent to
- Start of treatment (week 1)
- Pairing for first generation (week 11)
- Selection for second generation (week 18)
- Pairing for second generation (week 28)
- Lactation for second generation (week 33)


For each dose level a sub-sample was extracted with acetone using soxhlet apparatus. After dilution with acetone, a suitable volume was evaporated to dryness (using RFE). The residues was dissolved in HPLC mobile phase, then analysed by HPLC-UV.

The mean concentrations determined within 0.8% and 3.5% below nominal concentrations confirmed the accuracy of formulation.

Duration of treatment / exposure:

Males and females of the P (F0) generation were treated for 10 weeks before pairing and throughout the study until termination

Animals of the F1 generation had access to the same diet as their parents throughout, but the F1 generation was deemed to formally start at approximately 4 weeks of age (week 1 of the F1 generation). F1 animals were treated from weaning to approximately 10 weeks before pairing and until termination when litters were weaned.

Frequency of treatment:

The test material was administered to the animals in their diet, which was available on an ad libitum basis. Males and females ofthe FO generation were treated for 10 weeks before pairing and throughout the study until termination. Animals of the F1 generation had access to the same diet as their parents throughout, but the F1 generation was deemedto formally start at approximately 4 weeks of age. They were treated from weaning for approximately 10 weeks before pairing, and until termination when litters were weaned.

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Age at mating of the mated animals in the study: 16 - 18 weeks

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

1 000 ppm

Remarks:

Basis: nominal in diet

Dose / conc.:

3 300 ppm

Remarks:

Basis: nominal in diet

Dose / conc.:

10 000 ppm

Remarks:

Basis: nominal in diet

No. of animals per sex per dose:

(F0): 32/sex/dose
(F1): 28/sex/dose

The FOgeneration, which comprised 32 males and 32 females in each group, received the treated diet for 10 weeks before pairing and throughout mating, gestation, littering and lactation. Offspring survival, growth and sexual maturation were evaluated. From the litters 28 male and 28 female offspring per group, were selected to form the FI generation. Both sexes received similar treated diets as their parents for a minimum of 10 weeks from selection, throughout pairing, gestation, littering and lactation. F2 offspring were monitored for survival and development until weaning.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Dietary concentrations of 1000, 3300 and 10000 ppm were selected in collaboration with the Sponsor based on results from a preliminary dietary study performed at Huntingdon Life Sciences (Report No. VCL315/990088).

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily, with a more detailed examination performed weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the day that treatment commenced (F0) or the formal start of the generation (F1), then weekly thereafter. F0 and F1 females were weighed on the same schedule until mating was detected and then on Days 0, 6, 13 and 20 after mating and on Days 1, 4, 7, 14 and 21 of lactation.

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was recorded on a cage basis two animals per cage for F0 and F1 males and females weekly before pairing for mating. Food consumption for females after mating was recorded daily on an individual basis on Days 0-5, 6-12 and 13-19 after mating.
Food consumption for F0 and F1 females was recorded for Days 1-3, 4-6, 7-13 and 14-20 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

For 22 days before pairing of P (F0) and F1 generations, daily vaginal smears were taken using cotton swabs, from all females and examined to establish the duration and regularity of the oestrus cycle.

Sperm parameters (parental animals):

Parameters examined in F0/F1 male parental generations:
Immediately after scheduled sacrifice
- testis and epididymis weight
- sperm motility
- sperm morphology
- sperm count in epididymides
- homogenisation-resistant spermaids

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Litters containing more than ten offspring were culled by random selection to ten where possible five males and five females on Day 4 of age.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
- number and sex of pups (at days 1, 4 and 21 of age),
- stillbirths,
- live births,
- postnatal mortality,
- presence of gross anomalies,
- weight gain (Days 1, 4, 7, 14, and 21 of age),
- physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed once the majority of litters had weaned and it had been established that further litters were not required.
- Maternal animals: All surviving animals that littered and reared offspring were killed on Day 28 of lactation after completion of post-weaning vaginal smears.
Females whose litters died before weaning were generally killed on their theoretical Day 28 after completion of vaginal smearing similar to the females with surviving litters. Females that failed to mate mated but were not pregnant or failed to litter were retained and killed on the same day as the first batch of females with litters for that generation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, abdominal and pelvic cavities and their viscera.
The external and cut surfaces of the organs and tissues were examined either before or after weighing as appropriate. The number of uterine implantation sites was recorded for the adult females. Abnormalities interactions and changes were noted the requisite organs weighed and the required tissue samples preserved in fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
Adrenal glands, Prostate ventral, Brain, Seminal vesicles and coagulating gland, Epididymides, Spleen, Kidneys, Testes, Liver, Uterus with cervix, Ovaries with oviduct, Pituitary.
Paired organs weighed separately.
The weight of these organs were expressed as a percentage of the bodyweight recorded immediately prior to necropsy for all adults surviving to scheduled terminal kill.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed were subjected to macroscopic postmortem examinations (external and internal examination).

GROSS NECROPSY
Unselected F1 offspring and F2 offspring were examined macroscopically for evidence of disease or adverse reaction to treatment and appropriate organs weighed and retained. Any abnormal tissues were also retained.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs were taken from 1 male and 1 female randomly selected from each litter after weaning, dissected free from adjacent fat and other tissue and the weight recorded: Brain, Spleen, Thymus.
Abnormalities, Seminal vesicles and coagulating gland, Brain, Spleen, Epididymides, Testes, Ovaries, Thymus, Oviduct, Uterus with cervix, Prostate ventral lobe, Vagina.

Statistics:

- Analysis of variance followed by an intergroup comparison with the Control were performed (Bodyweights and bodyweight change, food consumption, litter data, sexual development data, seminology data, organ weights and histopathological findings).
- Homogeneity of variance using Bartlett's test (adult organ weights and weekly bodyweight change for the parental animals)
- Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparison otherwise a Dunnett's test was used. Intergroup differences in macroscopic pathology and histopathology waere assessed using Fisher's exact test.
- For bodweight and food consumption data during gestation and lactation, litter data, sexual development data and offspring organ weights, the statistical analysis was performed using an in-house programme. Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance), followed by Williams' test or non parametric tests (Kruskal Wallis, Hollander and Wolfe) followed by Shirley's test were used as appropriate..
- Where 75% or more of the values for a given variable were the sam,e a Fisher's exact test was used
Significant (p<0.05) inter group differences from the Control were reported.

Reproductive indices:

Percentage mating=(Animals mated/Animals paired) x 100
Conception rate=(Animals pregnant or siring a pregnancy/Animals mated) x 100
Fertility index=(Animals pregnant or siring a pregnancy/Animals paired) x 100
Gestation index=(Number of live litters born/Number pregnant) x 100

Offspring viability indices:

Post implantation survival index=(Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index=(Total number of live offspring on Day 1/Total number of offspring born) x 100
Viability index=(Number of live offspring on Day 4 of age/Number of live offspring on Day 1 of age) x 100
Lactation index=(Number of live offspring on Day of examination/Number of live offspring at Day 4 after culling) x 100
Sex-ratio=(Number of males in litter/Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were seen that were considered associated with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no unscheduled deaths amongst the males. There were two mortalities among females, both in the Control group: Female was killed for reasons of animal welfare during Week 14 (Day 1 of lactation). The animal had developed a mass on the right upper ventral thorax; reduced body temperature, piloerection, pallor and blood discharge from the vagina had been apparent on the day of sacrifice.
Another female was found dead during Week 15 (Day 7 of lactation) having shown underactivity, piloerection, pallor, red discharge from vagina, pale eyes and hunched posture during Week 14 and brown staining on the left lower jaw, muzzle and forelimbs.

Thirteen other females died due to natural causes or humane sacrifice. The nature and distribution of these deaths did not suggest an effect of treatment. The deaths were
therefore considered coincidental.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The bodyweights and bodyweight changes for the FO males were unaffected by treatment with Benzoflex® 9-88.There was no effect of treatment at 10000 ppm on bodyweight change during the first two Weeks of the pre-mating phase. However during Weeks 3-10 of the pre-mating phase, bodyweight change at 10000 ppm was marginally but significantly lower than in Controls; overall gain during Weeks 1-10 was 7% lower than in Controls. There was no obvious effect on weight change during gestation although absolute bodyweight remained lower than the concurrent control value. This difference persisted immediately after birth with Day 1 post partum bodyweight lower than in Controls and weight gain during Days 1-4 of lactation noticeably lower than in Controls (although the difference did not attain statistical significance). Weight change during Days 4-14 was not adversely affected by treatment. During Days 14-21, however, females did not show the usual pattern of late lactation weight loss (as seen in the Controls) but showed overall mean weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects on food consumption in the pre-mating phase.Throughout the pre-mating, gestation and lactation phases all groups of females consumed
comparable amounts of food.

Food efficiency:

no effects observed

Description (incidence and severity):

The food conversion efficiencies for the males and females were comparable to the Control values at all dietary concentrations during the pre-pairing period. This reflected the fact that there were no marked effects on bodyweight performance and food consumption through this period.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of the organs and tissues taken from the FO males and females did not reveal any findings that were considered to be related to treatment with Benzoflex" 9-88. In the vagina, there was a higher incidence of epithelial keratinisation among females which received 10000 ppm Benzoflex® 9-88 compared with Controls, but this was not considered to be of any toxicological significance. A total of three neoplasms were seen, all in females. There was an endometrial polyp in the uterus of an animal which received 10000 ppm, an adenocarcinoma in the caudal mammary gland of an animal which received 3300 ppm and an adenoma in the cranial mammary gland of a Control.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Vaginal smears taken post-weaning to provide evidence of return to oestrus cycling after lactation showed that there was a higher proportion of females in oestrus on the day of terminal kill in groups treated with Benzoflex" 9-88 compared with Controls. This may be reflected in the increased incidence of vaginal epithelial keratinisation detected by light microscopy in the 10000 ppm group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Quantitative (CASA) assessment of the sperm parameters: motility, progressive motility, sperm count, homogenisation resistant spermatids and visual assessment of sperm morphology gave no
cause for concern and appeared to be unaffected by treatment with Benzoflex" 9-88.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility, as assessed by pre-coital interval, percentage mating, conception rate and fertility index, were virtually identical for all groups. There was no indication of any adverse effects at any treatment level

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
F0
The general condition of F0 males and females was considered to be satisfactory throughout the study and no clinical signs considered to be associated with treatment were observed.
All males survived to termination.

F1
The general condition of F1 males and females was considered to be satisfactory throughout the study and no clinical signs considered to be associated with treatment were observed.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
F0
Males: Bodyweight and bodyweight changes were unaffected by treatment.
There were no adverse effects on food consumption in the pre-mating phase.

Females:
At 10000ppm, during the pre-mating phase, no effect was observed during the first 2 weeks, but a significant reduction was recorded during weeks 3-10. No effect was observed during gestation.
At 3300 and 1000 ppm, during the pre-mating phase, gestation and lactation, to weaning of the F1 litters, bodyweight and bodyweight change was comparable with Controls.
There were no adverse effects on food consumption in the pre-mating, gestation and lactation phases.

F1
Males: Mean body weights at the start of the generation were essentially comparable although the lowest value was recorded at 10000ppm reflecting the trend noted at D21 of age. Overall weight change during week 0-15 was significantly lower than in controls.
At 1000 and 3300ppm, overall bodyweight gain was slightly lower than that of controls, but as it was not dosage related and as there were no differences in either bodyweight or bodyweight change, it was considered to be not treatment related.
The amounts of food consumed during the first week of the pre-mating period were similar in all groups suggesting no apparent effect of treatment. During weeks 2-10 however, there was a tendency for marginally lower intake in groups treated with DPGDB compared with controls, reflecting the marginally lower asolute bodyweight compared to controls.

Females:
Bodyweight or bodyweight changes were comparable in all groups before mating and during gestation.
As in F0 generation, at 10000 ppm weights on Day 1 of lactation were slightly lower than in controls and weight gain during Days 1-4 was noticeably lower than in controls, although the difference did not attain significance.
At 1000 and 3300ppm, the pattern of weight change during lactation was comparable to controls.
During the period before mating and throughout gestation and lactation there were no obvious effects of DPGDB on food consumption.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
F0
Exposure levels in excess of 500 mg/kg/day were achieved at 10000 ppm throughout the 10 week pre mating period. The fluctuations during gestation and lactation were in line with expectations and were related to changes in the physiological demands on the parent females during these periods.
During lactation at the period of peak demand on the dam Days 4-13 the intake of the females was in excess of 1500 mg/kg/day at 10000 ppm.

F1
Exposure levels well in excess of 500 mg/kg/day were achieved at 10000 ppm prior to pairing and with a mean intake approaching 1000mg/kg/d in males and exceeding this in females.
The fluctuations during gestation and lactation were in line with expectations and were related to changes in the physiological demands on the parent females during these periods. During lactation at the period of peak demand on the dam Day 7-13 the intake of the females was in excess of 2000 mg/kg/day at 10000 ppm.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
F0
There was no adverse effect of treatment with DPGDB on oestrous cycles at any dietary inclusion level.

F1
There was no adverse effect of treatment with DPGDB on oestrous cycles at any dietary inclusion level.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0
Motility, progressive motility, sperm count, homogenisation resistant spermatids and visual assessment of sperm morphology gave no concern and appeared to be unaffected by DPGDB.

F1
The numbers of motile and progressively motile sperm (from the vas deferens) and the numbers of caudal epididymal sperm and testicular spermatids were similar in all groups. In addition, assessment of sperm morphology from a vas deferens sample suggested that DGPDB had no adverse effects upon sperm maturation.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0
Precoital interval and mating performance
Mating performance and fertility as assessed by precoital interval, percentage mating, conception rate and fertility index were virtually identical for all groups. There was no indication of any adverse effects at any treatment level.

Gestation length gestation index and parturition
Gestation length and gestation index showed no adverse effect of treatment; all females had gestation lengths within the expected range of 22 to 23 days. No problem was evident during parturition process that was considered to be related to treatment.

F1
Pre coital interval and mating performance
The mating performance and fertility of the F1 animals did not suggest any adverse effects at any treatment levels.

Gestation length gestation index and parturition
Gestation length and gestation index showed no adverse effect of treatment; all females had gestation lengths within the expected range of 22 to 23 days.


ORGAN WEIGHTS (PARENTAL ANIMALS)
F0
Among F0 males, terminal bodyweight was comparable in all groups. Absolute and relative weights of the adrenal glands were marginally but significantly higher than in controls at 10000 and 3300ppm. There were no other significant differences in absolute or relative organ weights.

Among F0 females, terminal bodyweight was 94%, 97%, and 93% of controls in the 1000, 3300 and 10000ppm groups, respectively. Absolute weight for the kidneys at 10000ppm was significantly lower than in controls but this was thought to reflect the difference in absolute terminal bodyweight. Relative weights for the adrenal glands and brain were significantly higher than in controls; the difference in brain weight is considered to be of non toxicological importance since absolute brain weight was similar to control, despite the slightly lower terminal bodyweight.

The higher absolute and relative weight for the adrenal glands observed at 10000 and 3300ppm are considered to be doubtful biological significance, as no treatment related findings were detected at microscopic examination.

F1
Among F1 males, terminal bodyweight was 95%, 97%, and 92% of controls in the 1000, 3300 and 10000ppm groups, respectively. Absolute weight for the kidneys at 10000ppm was significantly lower than in controls but this was thought to reflect the difference in absolute terminal bodyweight. Relative weights for the libver and brain wer significantly higher than in controls; the differences are considered to be of doubtful toxicological importance since absolute weights were similar to controls.

Relative weights for the adrenal glands and brain were significantly higher than in controls; the difference in brain weight is considered to be of non toxicological importance since absolute brain weight was similar to control, despite the slightly lower terminal bodyweight.


GROSS PATHOLOGY (PARENTAL ANIMALS)
F0
There were no macroscopic abnormalities detected at necropsy of the F0 males of females that were considered to be as a result of treatment with DPGDB.

F1
Macroscopic findings for the males and females in the treated groups were similar to the controls.


HISTOPATHOLOGY (PARENTAL ANIMALS)
F0
Microscopic examination of the organs and tissues taken from F0 males and females did not reveal any findings that were considered to be related to exposure to DPGDB.
In the vagina, there was a higher incidence of epithelial keratinisation among females which received 10000 DPGDB compared with controls, but this was not considered to be of toxicological significance.

Oestrous cycle at termination (parental F0 animals)
Vaginal smears taken post-weaning to provide evidence of return to oestrus cycling after lactation showed that there were a higher percentage of females in oestrus on the days of terminal kill in groups treated by DPGDB compared with controls.
This may be reflected in the increased incidence of vaginal epithelial keratinisation detected by light microscopy in the 10000ppm group.


F1
There were no microscopic findings that were considered to be related to treatment with DPGDB.

Oestrous cycle at termination (parental F1 animals)
Vaginal smears assessed for parental females for approximately one week after weaning (Days 22 to 28 of lactation) demonstrated that DPGDB did not appear to affect the F1 females ability to restore oestrous cycle within the expected period after weaning.

Ovarian primordial follicle counts F1 females
DPGDB had no apparent effect on the primordial follicle populations.

Key result

Dose descriptor:

NOEL

Effect level:

10 000 ppm (nominal)

Sex:

male/female

Remarks on result:

other: Generation: F1 parents (migrated information)

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of the F1 offspring in all groups was satisfactory and showed no apparent adverse responses to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The general condition of Fl animals was satisfactory throughout. There were two unscheduled deaths but these were not considered to be related to treatment. Female in the Control group was killed for reasons of animal welfare during Week 17 (Day 23 of lactation). The animal had thin build with a swollen area on the left mammary area with dark skin on the swollen area and, hunched posture.Ten females had total litter loss either due to natural causes or humane sacrifice. The number and distribution of these litter deaths did not suggest an effect of treatment and they were considered typical of the inherent pattern of litter deaths previously recorded in this laboratory with this strain of rat.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males : During Weeks 3-15 at 10000 ppm the weight change was slightly but consistently lower than in Controls; overall weight change during Weeks 0-15 was significantly lower than in Controls. At both 3300 and 1000 ppm, overall bodyweight gain was slightly lower than that of Control animals but this was not dosage related and there were no differences in either bodyweight or bodyweight change that were considered to be conclusively attributable to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The amounts of food consumed during the first week of the pre-mating period were similar in all groups suggesting no apparent effect of treatment. During Weeks 2-10 however, there was a tendency for marginally lower intake in groups treated with Benzoflex'" 9-88 compared with Controls and this difference was thought to reflect the marginally lower absolute bodyweights compared with Controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Among F1 males, terminal bodyweight was 95%, 97% and 92% of Controls in the 1000, 3300 and 10000 ppm groups respectively. Absolute kidney weight at 10000 ppm was significantly lower than in Controls but this was thought to reflect the difference in absolute terminal bodyweight since the bodyweight relative value was similar to Controls. Bodyweight relative weights for the liver and brain were significantly higher than in Controls; the differences are considered to be of doubtful toxicological importance since absolute weights were similar to Controls.
Among F1 females, terminal bodyweight was 97%, 99% and 95% of Controls in the 1000, 3300 and 10000 ppm groups respectively. Absolute and bodyweight relative kidney weights at 10000 ppm were significantly lower than in Controls. Absolute ovary plus oviduct weights at 10000 and 3300 ppm were significantly lower than in Controls but the differences were largely thought to reflect slight inter-group differences in bodyweight since there was no significant difference for the bodyweight relative weight at 10000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings for the males and females in the treated groups were similar to the Controls

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Macroscopic findings for the males and females in the treated groups were similar to the Controls.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One neoplasm, an adenocarcinoma of the cranial mammary gland, was seen in a female which
received 3300 ppm.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The age of attainment of vaginal opening among Fl females in groups treated with Benzoflex" 9-88 was slightly delayed compared with Controls but the differences did not show a relationship to dietary levels of Benzoflex® 9-88 and did not attain statistical significance. The slight delay in vaginal opening is thought to reflect the slightly lower absolute bodyweights at a given point in time among groups treated with Benzoflex" 9-88 since bodyweight was virtually identical between groups at the actual time of sexual maturation; no direct effect on sexual maturation was indicated.

The occurrence and regularity of oestrous cycles were considered to be unaffected by treatment with Benzoflex" 9-88 at any dietary concentration

Benzoflex" 9-88 had no apparent effect on the primordial follicle populations in any group tested
when compared to the Control values

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The timing of onset and completion of balano-preputial separation for the F1 males showed no adverse response to treatment and the bodyweight was comparable between groups at the time of completion of sexual maturation.

The numbers of motile and progressively motile sperm (from the vas deferens) and the numbers of caudal epididymal sperm and testicular spermatids were similar in all groups. In addition, assessment of sperm morphology from a vas deferens sample suggested that Benzoflex'" 9-88 had no adverse effects upon sperm maturation.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of animals failed to show evidence of mating but the number and distribution of these did not suggest an association with treatment with Benzoflex® 9-88. Thus, the mating
performance and fertility of the Fl animals did not suggest any adverse effects at any treatment level with both males and females at 10000 ppm comparing favourably with their Control counterparts.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The general condition of Fl animals was satisfactory throughout. There were two unscheduled deaths but these were not considered to be related to treatment. Male receiving 1000 ppm was found dead during Week 14 of the Fl generation. No cause for death was established. Female (one) in the Control group was killed for reasons of animal welfare during Week 17 (Day 23
of lactation). The animal had thin build with a swollen area on the left mammary area with dark skin on the swollen area and, hunched posture.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For both sexes, offspring bodyweights at birth were similar in all groups. Subsequent bodyweight change of both sexes to Day 14 of age was unaffected by treatment. Bodyweight change of male and female offspring in the 10000 ppm group was slightly lower than in Controls and marginally lower in the 1000 and 3300ppm groups during Days 14-21 of age, perhaps suggesting that the effect on weight gain was linked to the transition to direct exposure to the test material as the offspring weaned onto solid diet at the same dietary inclusion levels as their parents. For males, there were no statistically significant differences in overall weight change during Days 1-21 of age; for females, overall weight change was significantly lower than in Controls.


Mean bodyweight at the start of the generation were essentially comparable although the lowest value was recorded at 10000 ppm reflecting the trend noted at Day 21 of age. Subsequently, weight change at 10000 ppm during the first 3 Weeks of the Fl generation was comparable to Controls. During Weeks 3-15 however, weight change was slightly but consistently lower than in Controls; overall weight change during Weeks 0-15 was significantly lower than in Controls.
At both 3300 and 1000 ppm, overall bodyweight gain was slightly lower than that of Control animals but this was not dosage related and there were no differences in either bodyweight or bodyweight change that were considered to be conclusively attributable to treatment.


Bodyweight and bodyweight changes were comparable in all groups before mating and during gestation. As in the FO generation, at 10000 ppm weights on Day 1 of lactation were slightly lower than in Controls and weight gain during Days 1-4 was noticeably lower than in Controls although the difference did not attain significance. Weight change during Days 4-14 was not adversely affected by treatment. During Days 14-21 females did not show the expected pattern of late lactation weight loss (as seen in the Controls) but showed overall mean weight gain. At 1000 and 3300 ppm, the pattern of weight change during lactation was comparable to Controls.

Description (incidence and severity):

During the period before mating and throughout gestation and lactation there were no obvious effects of Benzoflex" 9-88 on food consumption. The amounts of food consumed during the first week of the pre-mating period were similar in all groups suggesting no apparent effect of treatment. During Weeks 2-10 however, there was a tendency for marginally lower intake in groups treated with Benzoflex'" 9-88 compared with Controls and this difference was thought to reflect the marginally lower absolute bodyweights compared with Controls.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiencies for the F1 males and females were essentially comparable to the Control values at all dietary concentrations. This reflected the fact that there were no marked effects on bodyweight performance and food consumption through this period.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The timing of onset and completion of balano-preputial separation for the F1 males showed no adverse response to treatment and the bodyweight was comparable between groups at the time of completion of sexual maturation. The age of attainment of vaginal opening among Fl females in groups treated with Benzoflex" 9-88 was slightly delayed compared with Controls but the differences did not show a relationship to dietary levels of Benzoflex® 9-88 and did not attain statistical significance. The slight delay in vaginal opening is thought to reflect the slightly lower absolute bodyweights at a given point in time among groups treated with Benzoflex" 9-88 since bodyweight was virtually identical between groups at the actual time of sexual maturation; no direct effect on sexual maturation was indicated.

sperm analysis

The numbers of motile and progressively motile sperm (from the vas deferens) and the numbers of caudal epididymal sperm and testicular spermatids were similar in all groups. In addition, assessment of sperm morphology from a vas deferens sample suggested that Benzoflex'" 9-88 had no adverse effects upon sperm maturation.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no obvious treatment related effects on the weights of the brain, spleen or thymus in the Fl offspring killed at 34 days of age. At 10000 ppm, the bodyweight relative weight for the brain for females was significantly higher than in Controls but this was considered to be of no toxicological importance as it reflected the fact that absolute brain weight was similar to Controls despite a significant reduction in absolute bodyweight.


Among F1 males, terminal bodyweight was 95%, 97% and 92% of Controls in the 1000, 3300 and 10000 ppm groups respectively. Absolute kidney weight at 10000 ppm was significantly lower than in Controls but this was thought to reflect the difference in absolute terminal bodyweight since the bodyweight relative value was similar to Controls. Bodyweight relative weights for the liver and brain were significantly higher than in Controls; the differences are considered to be of doubtful toxicological importance since absolute weights were similar to Controls.
Among F1 females, terminal bodyweight was 97%, 99% and 95% of Controls in the 1000, 3300 and 10000 ppm groups respectively. Absolute and bodyweight relative kidney weights at 10000 ppm were significantly lower than in Controls. Absolute ovary plus oviduct weights at 10000 and 3300 ppm were significantly lower than in Controls but the differences were largely thought to reflect slight inter-group differences in bodyweight since there was no significant difference for the bodyweight relative weight at 10000 ppm.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings for the males and females in the treated groups were similar to the Controls

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no microscopic findings that were considered to be related to treatment with Benzoflex® 9-88.One neoplasm, an adenocarcinoma of the cranial mammary gland, was seen in a female which received 3300 ppm

Other effects:

no effects observed

Description (incidence and severity):

Oestrous cycles: The occurrence and regularity of oestrous cycles were considered to be unaffected by treatment with Benzoflex" 9-88 at any dietary concentration.
Vaginal smears, taken on Day 22 to Day 28 of lactation, demonstrated that Benzoflex® 9-88 did not appear to affect the F1 females ability to restore oestrous cyclicity within the expected period after weaning.

Pre-coital interval and mating performance: A small number of animals failed to show evidence of mating but the number and distribution of these did not suggest an association with treatment with Benzoflex® 9-88. Thus, the mating performance and fertility of the Fl animals did not suggest any adverse effects at any treatment level with both males and females at 10000 ppm comparing favourably with their Control counterparts.

Gestation length, gestation index and parturition
The length of the gestation phase was 22 to 23 days for all females in all groups and gestation index showed no adverse effect of treatment. The process of parturition was successfully completed for the majority of the females showing no obvious adverse effects of treatment.

Ovarian primordial follicle counts Fl females
Benzoflex" 9-88 had no apparent effect on the primordial follicle populations in any group tested when compared to the Control values.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

VIABILITY (OFFSPRING)
F1
The number of uterine implantation sites recorded at termination, litter size at birth, survival of offspring to litter standardisation on Day 4 and subsequent survival to weaning did not indicate any adverse effects of treatment.

F2
At 10000 and 3300 ppm the mean number of uterine implantation sites and total litter size at birth were slightly lower than in controls. However, it didn’t attain statistical significance
Then mean number of implantations and total litter size at birth at 1000ppm were comparable to controls.
Survival of offspring prior to litter standardisation on Day 4 and subsequent survival at Day 21 of age did not suggest any adverse effects of treatment.


CLINICAL SIGNS (OFFSPRING)
F1
The general condition of the offspring was similar in all groups and showed no adverse responses to treatment of the F0 parents.

F2
There was no indication that the general condition of offspring had been adversely influenced by the treatment the parental female had received.


BODY WEIGHT (OFFSPRING)
F1
For both sexes offspring bodyweights at birth were similar in all groups.
Subsequent bodyweight change of both sexes to Day 14 of age was unaffected by treatment. Bodyweight change of male and female offspring in the 10000ppm group was slightly lower than in Controls and marginally lower in the 1000 and 3300 ppm groups during Days 14-21 of age.

F2
Absolute and relative bodyweights of the brain and thymus and the F2 generation were comparable in all groups, indicating no adverse effect of treatment.


SEXUAL MATURATION (OFFSPRING)
F1
The timing of onset and completion of balano-preputial separation for the F1 males showed no adverse response to treatment and the bodyweight was comparable between all groups at the time of completion of sexual maturation.

F2
Sex ration from Day 1 to 21 after birth, did show some slight inter-group variability, but it was not considered as treatment related.


ORGAN WEIGHTS (OFFSPRING)
F1
There were no obvious treatment related effects on the weights of the brain, spleen or thymus in the F1 offspring killed at 34 days of age. At 10000ppm the relative weight for the brain for females was significantly higher than in controls but this was considered to be of toxicological importance, as it reflected the fact that absolute brain weight was similar to controls, despite a reduction in absolute bodyweight.

F2
Absolute and relative weight of the brain and thymus of the F2 generation were comparable in all groups, indicating no adverse effect of treatment.
Absolute and relative weight of the spleen for males and females at 10000ppm were significantly lower than in controls.


GROSS PATHOLOGY (OFFSPRING)
F1
Macroscopic examination of offspring dying before weaning or unselected offspring killed at weaning, after selection of F1 generation, did not reveal any findings considered to be related to treatment with DPGDB. Pups commonly had no milk in the stomach reflecting possible lack of maternal care. This finding is frequently observed in offspring that die at such an early age.

F2
Macroscopic examination of offspring dying before weaning did not reveal any findings considered to be related to treatment with DPGDB.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

viability

clinical signs

gross pathology

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of the F2 offspring in all groups was satisfactory and showed no apparent adverse responses to treatment.

Dermal irritation (if dermal study):

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

Survival of offspring prior to litter standardisation on Day 4 and subsequent survival to Day 21 of age did not suggest any adverse effects of treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweights of the F2 offspring at birth were comparable in all groups. There was no effect of treatment on bodyweight gain of male and females during Days 1-14 of age. There was no conclusive effect of treatment on weight gain during Days 14-21 of age although it was noted that the lowest gain during this period occurred, as in the first generation, at 10000 ppm. Overall there was no relationship to dietary concentration and there were no significant differences in overall weight gain during Days 1-21 of age.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute and bodyweight relative weights of the brain and thymus of the F2 offspring were comparable in all groups, indicating no adverse effect of treatment. Absolute and bodyweight relative weights of the spleen for males and females at 10000 ppm were significantly lower than in Controls.

Findings for F2 progeny

Bodyweight relative spleen weights (% bodyweight) on Day 21 of age 0 1000 ppm 3300 ppm 10000 ppm
Males 0.4609 0.4102 0.4357 0.3736
Females 0.4802 0.4436 0.4654 0.4081

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of F2 offspring dying before weaning or at scheduled termination (Day 21 of age) did not reveal any findings considered to be related to treatment with Benzoflex® 9-88. Most offspring dying before weaning had no milk in the stomach; indicating, perhaps, a lack of maternal care, predominantly in litters showing total pup loss. Among offspring examined at terminal examination on Day 21 of age, no macroscopic abnormalities of the spleen were detected.

Histopathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of F2 offspring dying before weaning or at scheduled termination (Day 21 of age) did not reveal any findings considered to be related to treatment with Benzoflex® 9-88. Most offspring dying before weaning had no milk in the stomach; indicating, perhaps, a lack of maternal care, predominantly in litters showing total pup loss. Among offspring examined at terminal examination on Day 21 of age, no macroscopic abnormalities of the spleen were detected.

Other effects:

no effects observed

Description (incidence and severity):

Uterine implantation sites, litter size and survival
At 10000 and 3300 ppm, the mean number of uterine implantation sites (recorded at termination) and total litter size at birth were slightly lower than in Controls. However, the differences did not show a relationship to dietary concentration of Benzoflex® 9-88 and did not attain statistical significance.

The mean number of implantations and total litter size at birth at 1000 ppm were comparable to Controls.
Survival of offspring prior to litter standardisation on Day 4 and subsequent survival to Day 21 of age did not suggest any adverse effects of treatment.

Sex ratios
Sex ratio, from Day 1 to Day 21 after birth, did show some slight inter-group variability, but the magnitude of this was such that it was not considered to be as a result of treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

10 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

viability

clinical signs

gross pathology

Reproductive effects observed:

not specified

Conclusions:

The evidence from this study suggested that a dietary concentration of dipropylene glycol dibenzoate at 10000 ppm should be considered as the No-Observed-Effect-Level (NOEL) for P (F0) and F1 parent animals. The No-Observed-Adverse-Effect-Level (NOAEL) for survival and growth of the offspring is considered to be 10000 ppm.

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 two generation study in rats was conducted to assess the effects on reproductive performance of the test material DPGDB. The study was conducted according to OECD and EPA test guidelines, and in compliance with GLP.

 

Dietary administration of DPGDB at concentrations of 1000, 3300 or 10000 ppm was generally well tolerated by the P (F0) and subsequent F1 parental animals and their respective progeny. Exposure to the test material was in line with expectation throughout both generations fluctuations reflected the different physiological status of the animals and were predictably highest for females during peak lactation and in young animals.  Bodyweight change of F1 females before paring and F1 males were slightly but significantly lower than in Controls.No adverse effects were seen on overall parental food consumption; food conversion efficiency calculated during the 10 week pre-mating phase was considered similar to controls for both generations.Oestrous cycle, mating performance, fertility and fecundity were similar in all groups. Gestation lengths and the parturition process were unaffected by treatment. Assessment of the terminal vaginal smears taken from F0 females revealed a higher incidence of females in oestrus in groups treated with DPGDB compared with controls. This finding was not apparent among F1 females and is considered to be of doubtful biological significance.

 

Litter parameters at birth of the F1 and F2 progeny and their survival to weaning showed no apparent detrimental effects of treatment with DPGDB. However, in both F1 and F2 offspring at 10000 ppm there was a slight reduction on weight gain during days 14-21 of age and this finding may be linked to the transition to direct exposure to the test material as the offspring weaned on to solid diet at the same dietary inclusion levels as their parents.

 

No treatment related findings were seen at microscopic examination of the F1 offspring not selected to form the next generation or the F2 offspring killed after weaning.Macropathology, histopathology assessment and sperm analysis for the F0 and F1 adults showed no adverse effects of treatment.

 

The only possible effect of treatment detected at assessment of organ weights from F1 and F2 offspring was significantly lower absolute and relative spleen weight among F2 males and females compared to controls. The toxicological significance if this finding is uncertain since it was not detected among F1 offspring or among F0/F1 adult animals.The evidence from this study suggested that a dietary concentration of DPGDB at 10000 ppm should be considered as the No Observed Adverse Effect Level (NOAEL) for P (F0) and F1 parent animals. The NOAEL for developing offspring is considered to be 3300 ppm. The No Observed Effect Level (NOEL) for reproductive parameters is considered to be 10000 ppm.

The evidence from this study suggested that a dietary concentration of DPGDB at 10000 ppm should be considered as the No-Observed-Effect-Level (NOEL) for F0 and F1 parent animals. The No-Observed-Adverse-Effect-Level (NOAEL) for survival and growth of the offspring is considered to be 10000 ppm.

Reference 3

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

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

Adequacy of study:

key study

Study period:

Not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across justification included in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (F0) animals : 2 weeks

- Basis for dose level selection : Benzoic acid concentrations were selected based on the results of a dose range finding (OECD 422-type; OECD 2016) study, supplemented by a 14-day palatability study in adult rats. In the palatability study, benzoic acid was generally well tolerated by both males and non-pregnant females at 7500, 11,500, 15,000 and 19,000 ppm in the diet. Based on these results, constant dietary concentrations of 11,500, 15,000, and 19,000 ppm were selected for the dose range-finding study. In the range-finding study, the maximum tolerated dose (MTD) for lactating females was exceeded for the 15,000 ppm and 19,000 ppm groups, with associated adverse effects observed including moribundity, mortality and adverse neuropathology (neuronal degeneration/necrosis in the hippocampus and amygdala) in the majority of females (10 of 12) in the 19,000 ppm groups. The neuropathological findings were considered the cause of death/moribundity. Similar observations have previously been noted following dietary benzoic acid administration at 30,000 ppm in Royal Wistar rats following 5 days of exposure (Kreis, 1967). Consequently, the dietary concentrations used in the current study were adjusted weekly for F0 and F1 females during lactation (and also during gestation), based on body weight and food consumption data from age-matched historical controls from previous multigenerational studies conducted at the testing facility, to adhere to target dose levels of 500, 750 and 1000 mg/kg bw/day. Similar weekly adjustments were also made in the dietary concentrations administered to F1 and F2 pups following weaning (PND 21–70) because animals at that stage of growth also show substantially greater food intake per kg of body weight than do adults.

- Inclusion/exclusion of extension of Cohort 1B : Included. F1 males and females assigned to Cohort 1B (optional reproductive assessments) were bred in a similar manner as the F0 adults, and females were allowed to rear their litters normally until weaning on PND 21, when 2 pups/sex/ litter were randomly selected to constitute the F2 generation.

- Termination time for F2 : PND 91

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B : Included. F1 animals designated for developmental neurotoxicity assessments were assigned to Cohorts 2A and 2B. Offspring were administered benzoic acid in the diet beginning at weaning and continuing until euthanasia Cohort 2A [PND 78, 8 weeks] and Cohort 2B [PND 22, 1 day].

In Cohort 2A, animals were evaluated for neurobehavior (auditory startle response, functional observational battery [FOB], locomotor activity and learning and memory [Biel maze] assessment) during the in-life period, and at termination (PND 78), brain measurements (weight, length, width) and
neuropathology, including microscopic morphometric measurements on homologous stained sections of the brain. Animals assigned to Cohort 2B were used for brain measurements (weight, length, width) and neuropathological evaluation of brain on PND 22.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Included. F1 animals designated for developmental immunotoxicity assessments were assigned to Cohort 3. Offspring were administered benzoic acid in the diet beginning at weaning and continuing until euthanasia: Cohort 3 [PND 59, 5.5 weeks]. Animals were evaluated for T-Cell Dependent Antibody Response (TDAR) at termination. In addition, 10 additional control rats/sex were assigned to Cohort 3A - a subset of Cohort 3 - which served as the positive control group. All animals in Cohort 3 were immunized with Sheep Red Blood Cells (sRBC) on PND 54.

- Route of administration : Oral dietary, based on the long history of use of Benzoic acid and its salts (sodium benzoate, potassium benzoate, and calcium benzoate) as food preservatives.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: Non-sibling male and female Sprague Dawley (Crl:CD(SD)) rats
approximately 10 weeks old at the initiation of benzoic acid administration. Number of animals are presented in Table 1 in the section 'any other information on materials and methods incl. tables'.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source not specified but most likely to be the Sponsor (American Beverage Association); Batch Number: STBG4672V
- Purity, including information on contaminants, isomers, etc.: >99% (meeting FCC and JECFA specifications for purity)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified in the publication
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Not specified in the publication

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

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

Non-sibling male and female Sprague Dawley (Crl:CD(SD)) rats approximately 10 weeks old at the initiation of benzoic acid administration. All animals were
received in good health and females were nulliparous and non-pregnant.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Raleigh (North Carolina, USA)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) approximately 10 wks
- Weight at study initiation: Not specified in the publication
- Fasting period before study: Not specified in the publication
- Housing: 2–3 rats/cage in solid-bottom cages containing heat-treated aspen bedding material
- Use of restrainers for preventing ingestion (if dermal): No
- Diet (e.g. ad libitum): certified rodent feed (PMI Nutrition International Rodent LabDiet® 5002) ad libitum throughout the study
- Water (e.g. ad libitum): Reverse osmosis-purified municipal water ad libitum throughout the study
- Acclimation period: Not specified in the publication

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–26°C
- Humidity (%): 30-70%
- Air changes (per hr): Not specified in the publication
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: Not specified in the publication

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- During periods of benzoic acid exposure, the basal feed was mixed with appropriate amounts of the test material for the treated groups. Benzoic acid was incorporated in the dry ground (meal) diet at concentrations of 0, 7,500, 11,500, and 15,000 ppm. F0 males were administered benzoic acid continuously in the diet for 2 weeks prior to mating and continuing until euthanasia (10–11 weeks). F0 females were administered benzoic acid continuously in the diet for 2 weeks prior to mating and continuing throughout mating (2 weeks), gestation (3 weeks), and lactation (3 weeks), until euthanasia (10–11 weeks). The offspring selected for the F1 generation were administered benzoic acid in the diet beginning at weaning and continuing until euthanasia (Cohort 1A [PND 91, 10 weeks], Cohort 2A [PND 78, 8 weeks], Cohort 2B [PND 22, 1 day], Cohort 3 [PND 59, 5.5 weeks], and Cohort 1B [until euthanasia following reproductive assessments]). The Cohort 1B offspring selected for the F2 generation were also administered benzoic acid in the diet beginning at weaning and continuing until euthanasia (PND 91, 10 weeks).

Dietary benzoic acid concentrations used in the study were adjusted weekly for F0 and F1 females during lactation (and also during gestation), based on body weight and food consumption data from age-matched historical controls from previous multigenerational studies conducted at the testing facility, to adhere to target dose levels of 500, 750 and 1000 mg/Kg bw/day. Similar weekly adjustments were also made in the dietary benzoic acid concentrations administered to F1 and F2
pups following weaning (PND 21–70) because animals at that stage of growth also show substantially greater food intake per kg of body weight than do adults.

- Storage temperature of food: Not specified in the publication

Details on mating procedure:

- M/F ratio per cage: 1:1 in the home cage of the male
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal copulatory plug or sperm in a vaginal lavage referred to as GD day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not specified in the publication
- After successful mating each pregnant female was caged (how): The dam and litter remained together until weaning on PND 21.
- Any other deviations from standard protocol: Not specified in the publication

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details on the methods used for analytical verification of benzoic acid concentration in feed were not specified in the publication.

Duration of treatment / exposure:

F0 (males): 10-11 weeks
F0 (females): 10-11 weeks
F1 (males & females): Cohort 1A: 10 weeks; Cohort 1B: 10 weeks; Cohort 2A: PND78/ 8 weeks; Cohort 2B: PND 22/ 1 day; Cohort 3: PND 59/ 5.5 weeks
F2 (males and females): F2 generation: PND91

Frequency of treatment:

continuously in diet

Details on study schedule:

- F1 parental animals not mated until 12 weeks after selected from the F1 litters.

- Selection of parents from F1 generation when pups were 13 weeks of age.

- Age at mating of the mated animals in the study: 10-13 weeks

Dose / conc.:

0 ppm

Remarks:

Control (Diet only)

Dose / conc.:

7 500 ppm

Remarks:

Equivalent to approximately 500 mg/Kg bw/day

Dose / conc.:

11 500 ppm

Remarks:

Equivalent to approximately 750 mg/Kg bw/day

Dose / conc.:

15 000 ppm

Remarks:

Equivalent to approximately 1000 mg/Kg bw/day

No. of animals per sex per dose:

F0: 30/sex/concentration
F1: Cohort 1A: 20/sex/concentration; Cohort 1B: 25/sex/concentration; Cohort 2A: 12/sex/concentration; Cohort 2B: 12/sex/concentration; Cohort 3: 10/sex/concentration; Cohort 3A (cyclophosphamide controls only): 10/sex/concentration

Control animals:

yes, concurrent no treatment

other: Control animals in Cohort 3A were exposed to cyclophosphamide, a known immunosuppressant, and served as positive controls for the T-cell antibody response assay (TDAR).

Details on study design:

- Dose selection rationale:
Benzoic acid concentrations were selected based on the results of a dose range finding (OECD 422-type; OECD 2016) study, supplemented by a 14-day palatability study in adult rats. In the palatability study, benzoic acid was generally well tolerated by both males and non-pregnant females at 7500, 11,500, 15,000 and 19,000 ppm in the diet. Based on these results, constant dietary concentrations of 11,500, 15,000, and 19,000 ppm were selected for the dose range-finding study. In the range-finding study, the maximum tolerated dose (MTD) for lactating females was exceeded for the 15,000 ppm and 19,000 ppm groups, with associated adverse effects observed including moribundity, mortality and adverse neuropathology (neuronal degeneration/necrosis in the hippocampus and amygdala) in the majority of females (10 of 12) in the 19,000 ppm groups. The neuropathological findings were considered the cause of death/moribundity. Similar observations have previously been noted following dietary benzoic acid administration at 30,000 ppm in Royal Wistar rats following 5 days of exposure (Kreis, 1967). Consequently, the dietary concentrations used in the current study were adjusted weekly for F0 and F1 females during lactation (and also during gestation), based on body weight and food consumption data from age-matched historical controls from previous multigenerational studies conducted at the testing facility, to adhere to target dose levels of 500, 750 and 1000 mg/kg bw/day. Similar weekly adjustments were also made in the dietary concentrations administered to F1 and F2 pups following weaning (PND 21–70) because animals at that stage of growth also show substantially greater food intake per kg of body weight than do adults.

- Rationale for animal assignment (if not random):
After acclimation to the testing facility, F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. On PND 21, randomly selected F1 pups were assigned to Cohorts 1, 2 and 3 such that not more than 1 pup/sex/litter was assigned to each cohort. For cohort 1B animals, on PND 21, 2 pups/sex/litter were randomly selected to constitute the F2 generation.

- Fasting period before blood sampling for clinical biochemistry: Not specified in the publication

Positive control:

Control animals in Cohort 3A were exposed to cyclophosphamide, a known immunosuppressant, and served as positive controls for the T-cell antibody response
assay (TDAR).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were monitored once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured weekly throughout the study, and twice weekly during gestation and lactation for mated females.

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: Yes
Food consumption was measured weekly throughout the study, and twice weekly during gestation and lactation for mated females. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported for each sex and group.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Mean benzoic acid intake (on a mg/Kg bw/day basis) was calculated and reported for each sex and group. Benzoic acid intake was calculated based on food consumed (g/Kg bw/day) and the appropriate concentration of benzoic acid in the food (ppm or mg/Kg).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

Beginning with the first day of test material administration, female (P0) oestrous cycles were monitored based on daily vaginal lavages (2 weeks prior to mating) and throughout the mating period, until evidence of mating (by the presence of a vaginal copulatory plug or sperm in a vaginal lavage). A terminal vaginal lavage sample was collected for all females to determine the stage of the oestrous cycle at the time of necropsy.

Sperm parameters (parental animals):

Parameters examined in all male parental generations: motility, concentration and morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce variability among the litters, ten pups from each litter, of equal sex distribution (where possible) were randomly selected on PND 4.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF REPRODUCTIVE TOXICITY:
F1 animals designated for reproductive toxicity assessments were assigned to Cohorts 1A and 1B.

In Cohort 1A, animals were evaluated for onset of oestrous and oestrous cyclicity during the in-life period (PND 75–91), splenic lymphocyte immunophenotyping (PND 91) and, similar to the parental (F0) generation, thyroid hormones, sperm parameters and clinical pathology, organ weights and histopathology at termination (PND 91). Onset of oestrus was monitored based on daily vaginal lavages beginning on the day of attainment of vaginal patency until cornified cells were observed in a lavage specimen. Spleens were processed for enumeration of live cell concentrations of Total, Helper and Cytotoxic T lymphocytes, B lymphocytes, NK and NK-T lymphocytes using Luna-FL™ cell counter and immunophenotyping using a BD FACSCanto™ II Flow Cytometer.

F1 animals assigned to Cohort 1B were mated to produce an F2 generation to evaluate the effects of benzoic acid on F1 pup reproductive behavior, parturition, litter parameters, offspring survival, growth and development until adulthood (PND 91). Maternal and litter parameters assessed for the bred females and their litters were similar to the parental (F0) generation.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
F1 animals designated for developmental neurotoxicity assessments were assigned to Cohorts 2A and 2B.

In Cohort 2A, animals were evaluated for neurobehavior (auditory startle response, functional observational battery [FOB], locomotor activity and learning and memory [Biel maze] assessment) during the in-life period, and at termination (PND 78), brain measurements (weight, length, width) and
neuropathology, including microscopic morphometric measurements on homologous stained sections of the brain. Brain morphometric measurements were performed only on samples from control and high-dose animals. Examination of low- and mid-dose animals was not conducted or warranted based on the lack of positive findings in the high-dose animals.

Auditory startle response was assessed on PND 24 using the MED Associates Startle Reflex System (St. Albans, VT). Sessions consisted of 50 trials each, with each trial consisting of 110–120 dB mixed-frequency noise burst stimulus, approximately 20 mins in duration. The intertrial interval was 8 s. Startle response measurements obtained were PEAK (peak response amplitude) and Tpeak (latency to PEAK). An FOB was conducted on PND 65, prior to the locomotor activity assessments, by trained technicians, blind to treatment group, and was counterbalanced across testing time, sex, and group. Animals were evaluated in the home cage, during handling, in an open field arena and observations included sensory, neuromuscular and physiological observations. Locomotor activity was monitored immediately following the FOB using the Kinder Scientific Motor Monitor System (Kinder Scientific Company, LLC, Poway, CA). Sessions were 60 min in duration. Total motor activity was evaluated as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of two or more consecutive photobeams). Lastly, a learning and memory assessment of the type normally conducted during the OECD (2007) Test Guideline 426 developmental neurotoxicity study was conducted for the same animals beginning on PND 66. Learning and memory was assessed using a water-filled 8-unit T-maze (Biel 1940). Animals were placed in the maze and were required to traverse the maze and escape by locating a submerged platform. The time to escape the maze and the number of errors were evaluated.

Animals assigned to Cohort 2B were used for brain measurements (weight, length, width) and neuropathological evaluation of brain on PND 22.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
F1 animals designated for developmental immunotoxicity assessments were assigned to Cohort 3.

Animals were evaluated for T-Cell Dependent Antibody Response (TDAR) at termination. In addition, 10 additional control rats/sex were assigned to Cohort 3A - a subset of Cohort 3 - which served as the positive control group. All animals in Cohort 3 were immunized with Sheep Red Blood Cells (sRBC) on PND 54. The primary IgM antibody response to sRBC, a T-dependent antigen, is an evaluation of humoral immunity, and cyclophosphamide, a known immune suppressant, was administered to all animals in the positive control group for 5 consecutive days (2.5 mg/Kg bw/day, PND 54–58) to demonstrate that the assay could detect suppression of the humoral immune response. The TDAR was conducted using a Rat Anti-sRBC IgM ELISA kit (Life Diagnostics, West Chester, PA) for the quantitation of rat anti-sRBC levels in serum samples.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (after the last litters in each generation were produced)
- Maternal animals: All surviving animals (post weaning on PND 21)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
At termination tissues and organs were collected for organ weights and full histopathological evaluation.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 91
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Not specified in the publication

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS

F1 Generation:

Cohort 1A: Spleens were processed for enumeration of live cell concentrations of Total, Helper and Cytotoxic T lymphocytes, B lymphocytes, NK and NK-T lymphocytes using Luna-FL™ cell counter and immunophenotyping using a BD FACSCanto™ II Flow Cytometer.

Cohort 1B: mated to produce an F2 generation, therefore no histopathogy conducted on this cohort.

Cohort 2A: brain measurements (weight, length, width) and neuropathology, including microscopic morphometric measurements on homologous stained sections of the brain. Brain morphometric measurements were performed on samples from control and high-dose animals; examination of low- and mid-dose animals was not conducted or warranted based on the lack of positive findings in the high-dose animals.

Cohort 2B: used for brain measurements (weight, length, width) and neuropathological evaluation of brain on PND 22.

Cohort 3: used to assess developmental immunotoxicity (T-Cell Dependent Antibody Response (TDAR) Assay).

F2 Generation:

Brains were collected, weighed and preserved for histopathological evaluation.

Statistics:

For information on statistics, please see the section 'Any other information on materials and methods, incl. tables'.

Reproductive indices:

Reproductive performance was summarized in terms of mating, fertility, conception and copulation indices

Offspring viability indices:

F1 and F2 generation - litter parameters and pup survival indices:
1) Implantation Sites
2) Pups Born
3) Unaccounted Sites
4) Live Litter Size (PND 0)
5) Males Per Litter (%)
6) Male Birth Weight (g, PND 1)
7) Female Birth Weight (g, PND 1)
8) Pup Survival (PND 0–4, % per litter)
9) Pup Survival (PND 4–21, % per litter)
10) Male Body Weight (g, PND 21)
11) Female Body Weight (g, PND 21)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed through the study period in the F0 generation male and female rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortality was observed through the study period in the F0 generation male and female rats.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related effects on body weight of male or female rats in the F0 generation were observed through the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related effects on food consumption of male or female rats in the F0 generation were observed through the study period.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency of male or female rats in the F0 generation was unaffected by treatment.

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology parameters evaluated in male and female rats in the F0 generation were unaffected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry parameters evaluated in male and female rats in the F0 generation were unaffected by treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

At termination, thyroid hormone (T4 and TSH) levels in F0 male and female rats were unaffected by treatment.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic observations at necropsy did not reveal any treatment-related findings.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed on F0 female estrous cyclicity.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed on F0 male sperm parameters (motility, concentration and morphology) evaluated.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performace remained unaffected by treatment.

At all doses administered, benzoic acid had no effects on survival in the F0 generation and no benzoic acid-related clinical signs nor any significant effects on male or female body weights or food consumption. There was no effect of dietary benzoic acid administration on F0 reproductive performance, female oestrous cyclicity or sperm parameters. At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F0 males and females and there were no treatment-related gross macroscopic or microscopic observations at necropsy or any effects on clinical pathology (hematology, serum chemistry, urinalysis, and bile acids) or organ weights.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no benzoic acid-related clinical signs observed in the F1 generation through the study period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of pups born and post-natal survival in the F1 generation were unaffected by treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Pup body weights and body weight gains across all groups were comparable to the control group during PND 1–14. Although lower pup body weight gains were noted for both males and females during PND 14–21, the reductions in absolute mean body weights were within the range of Charles River Ashland historical control data for reproduction studies and were non-dose responsive, therefore considered unrelated to benzoic acid administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no significant effects on food consumption observed.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No effects on haematological parameters were observed in F1 animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No effects on clinical chemistry parameters were observed in F1 animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No effects were observed in F1 animals.

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Exposure to benzoic acid had no effect on pup pre-weaning or post-weaning developmental landmarks.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Exposure to benzoic acid had no effect on pup pre-weaning or post-weaning developmental landmarks.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights of F1 animals remained unaffected by treatment with benzoic acid.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any remarkable findings.

Histopathological findings:

no effects observed

Description (incidence and severity):

No treatment-related microscopic findings were observed during the histopathological evaluation.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F1 males and females. Minor variations in T4 and TSH levels were not considered treatment-related because they were not noted in a dose responsive manner, were not generally statistically significant, or were observed in a direction that would be generally not be considered toxicologically relevant. These minor variations also fell within the range of levels noted for historical controls.

There was no effect of dietary benzoic acid administration on F1 reproductive performance, female oestrous cyclicity, time to first oestrus, female primordial follicle counts or sperm parameters. Higher mean cauda epididymal weights were noted in all treated groups when compared to controls. Correspondingly, statistically significantly lower mean cauda epididymal sperm concentrations (millions/gram tissue) were noted in all benzoic acid-treated groups compared to the control
Group. However, these differences were not considered treatment-related but ascribed to the atypically low mean cauda epididymal weight and consequently higher mean cauda epididymal sperm concentration observed for the control group, which was attributed to 3 (of 20) males in the control group with atypically low values for cauda epididymal weight (0.0795–0.1502 g versus a historical control mean ± SD of 0.2942 ± 0.032 g).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic findings or effects on gross brain measurements (weight, width, length) in F1 pups. There were also no treatment-related effects on microscopic morphometric measurements S1-S6 in the brains of young adult pups. A single measurement (Mean S2, Females – thickness of the parietal cortex) was significantly lower in 15,000 ppm group (1000 mg/Kg bw/day) females than in control group females. However, the value was within the range of the historical control data and thus, in the absence of any correlating changes in males, or changes in gross brain measurements, or alterations in neurobehavioral responses, this single change was considered to be an incidental statistical flag (Garman et al., 2016). In the neurobehavioral assessment, there were no effects of treatment on auditory startle responsiveness (peak response or time to peak response on PND 24), functional observational battery (FOB) and locomotor activity (ambulatory counts or total counts on PND 65) or learning and memory assessments on PND 66.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

There were no benzoic acid-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the TDAR Assay. The positive control, cyclophosphamide (CPS) produced the expected significant decrease in anti-sRBC-IgM in response to immunization with sRBC. Individual responses for all treated animals were within the range of control values with the exception of two 15,000 ppm (1000 mg/Kg bw/day) females that were identified as high responders. The mean value for the 15,000 ppm group was not statistically significantly different from the control group. Furthermore, the TDAR is designed to assess a decrease in the T cell-mediated response to the antigen, sRBC, by detecting anti-sRBC-IgM. Because a higher response to immunization is not toxicologically relevant, the isolated increase in 2 females was considered incidental. In addition, there was no effect on splenic lymphocyte populations (Total T, helper T and cytotoxic T lymphocytes, B lymphocytes, NK and NK-T cells) following exposure to benzoic acid at the doses administered.

Cohort 1: There were no benzoic acid-related clinical signs observed in the F1 generation through the study period and the mean number of pups born and post-natal survival in the F1 generation was unaffected by treatment. Pup body weights and body weight gains across all groups were comparable to the control group during PND 1–14. Although lower pup body weight gains were noted for both males and females during PND 14–21, the reductions in absolute mean body weights were within the range of the laboratory’s historical control data for reproduction studies and were non-dose responsive, therefore considered unrelated to benzoic acid administration. There were no significant effects on food consumption observed and haematological and clinical chemistry parameters were unaffected post exposure to the test material. Exposure to benzoic acid had no effect on pup pre-weaning or post-weaning developmental landmarks (anogenital distance, nipple retention) and gross necropsy did not reveal any remarkable findings. Organ weights of F1 animals were also unaffected by treatment and no treatment-related microscopic findings were observed during the histopathological evaluation.

At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F1 males and females. There was no effect of dietary benzoic acid administration on F1 reproductive performance, female oestrous cyclicity, time to first oestrus, female primordial follicle counts or sperm parameters. Higher mean cauda epididymal weights were observed in all treated groups when compared to the controls. Correspondingly, statistically significantly lower mean cauda epididymal sperm concentrations (millions/gram tissue) were noted in all benzoic acid-treated groups compared to the control group. However, these differences were not considered treatment-related but ascribed to the atypically low mean cauda epididymal weight and consequently higher mean cauda epididymal sperm concentration observed for the control group, which was attributed to 3 (of 20) males in the control group with atypically low values for cauda epididymal weight (0.0795–0.1502 g versus a historical control mean ± SD of 0.2942 ± 0.032 g).

Cohort 2: There were no treatment-related macroscopic findings or effects on gross brain measurements (weight, width, length) in F1 pups. There were also no treatment-related effects on microscopic morphometric measurements S1-S6 in the brains of young adult pups. In the neurobehavioral assessment, there were no effects of treatment on auditory startle responsiveness (peak response or time to peak response on PND 24), functional observational battery (FOB) and locomotor activity (ambulatory counts or total counts on PND 65) or learning and memory assessments on PND 66.

Cohort 3: There were no benzoic acid-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the TDAR Assay. The positive control (Cohort 3A), cyclophosphamide (CPS) produced the expected significant decrease in anti-sRBC-IgM in response to immunization with sRBC

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

developmental neurotoxicity

developmental immunotoxicity

other: Please see 'remarks'

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No benzoic acid-related clinical signs of toxicity were observed in the F2 generation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No benzoic acid-related effects on body weights were observed in the F2 generation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No benzoic acid-related effects on food consumption were observed in the F2 generation.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Exposure to benzoic acid had no effect on absolute or relative (to final body weight) brain weights in any group of males or females in the F2 generation.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The mean number of pups born, live litter size, offspring sex ratio and postnatal survival in the F1 generation were unaffected by benzoic acid. Pup body weights and body weight gains were unaffected by benzoic acid during the preweaning period and there were no treatment-related effects on weanling organ weights. For pups selected to constitute the F2 generation, there were no benzoic acid related clinical signs nor any significant effects on male or female body weights or food consumption, and lastly, exposure to benzoic acid had no effect on absolute or relative (to final body weight) brain weights in any group of males or females in the F2 generation.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Analytical testing confirmed stability of benzoic acid and homogeneity in feed. For the duration of the study, in all 3 generations evaluated (F0, F1 and F2), mean benzoic acid intake was maintained at dose levels within 10% of target levels (500, 750 and 1000 mg/Kg bw/day). Exceptions for both F0 and F1 males were noted during the post mating period where mean food consumption, and consequently test substance intake was 19–22% and 27–31% lower, respectively, than target (see Table 2). Reduced food consumption following separation from the females is not uncommon in male rodents. Theadjustments made to female dietary concentrations during gestation and lactation and for F1 and F2 pups during the postweaning period successfully allowed for maintenance of target dose levels.

Table 2. Mean benzoic acid intake (mg/Kg bw/day) – F0, F1 and F2 generations
	Mean Benzoic Acid Intake (mg/Kg bw/day)			% Difference from Target Dose levels
Dietary Concentration (ppm) Groups	7500	11500	15000	7500	11500	15000
Target Dose Level (mg/kg bw/day)	500	750	1000	500	750	1000
F0 Males
Prior to Mating	526	821	1069	5.2%	9.5%	6.9%
After Mating	389	601	807	-22.2%	-19.9%	-19.3%
F0 Females
Prior to Mating	526	821	1069	5.2%	9.5%	6.9%
Gestation	484	721	965	-3.2%	-3.9%	-3.5%
Lactation	512	767	1020	2.4%	2.3%	2.0%
F1 Males
Prior to Mating (PND 21–98)^a	499	747	987	-0.2%	-0.4%	-1.3%
After Mating (Cohort 1B Only)	349	542	689	-30.2%	-27.7%	-31.1%
F1 Females
Prior to Mating (PND 21–98)^a	535	790	1024	7.0%	5.3%	2.4%
Gestation (Cohort 1B Only)	499	712	957	-0.2%	-5.1%	-4.3%
Lactation (Cohort 1B Only)	512	771	1020	2.4%	2.8%	2.0%
F2 Males
Post Weaning (PND 21–91)	491	729	969	-1.8%	-2.8%	-3.1%
F2 Females
Post Weaning (PND 21–91)	504	737	988	0.8%	-1.7%	-1.2%

^a F1 Generation data (prior to mating) includes means of data for all cohorts, until the time of respective euthanasia

Note: Data shown are means of weekly means; data not subjected to statistical analysis.

Table 3. F0 Generation – Serum Total Thyroxine (T4, pg/mL) and Thyroid Stimulating Hormone (TSH, ng/mL) in Adult Male and Female Rats
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^* Mean (±2 SD)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F0 Males
Total T4	31630 ± 3996	36550 ± 6820	31530 ± 6512	31420 ± 4398	46640 (24743 - 68557)
TSH	7.1 ± 2.2	10 ± 4.1	10.3 ± 5.7	10.9 ± 3.9	9.6 (2.6 – 16.5)
F0 Females
Total T4	25800 ± 5337	29980 ± 8491	27840 ± 7180	27710 ± 7441	35144 (19639-50650)
TSH	7.3 ± 2.6	7.5 ± 4.1	6 ± 2.8	6.8 ± 4.2	5.0 (0.19 – 9.8)

* Charles River Ashland historical control data (version 2020.02).

Mean values are presented ±standard deviation (n = 10/sex/group).

None statistically significantly different from the control group.

Table 4. F0 generation – reproductive performance and male sperm parameters
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^*Mean (Min - Max)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F0 Males and Females
Mating index (%)	100.0	93.3	100.0	100.0	98.5 (93.3 – 100.0)
Fertility index (%)	96.7	93.3	100.0	96.7	93.9 (80.0 – 100.0)
Copulation / Conception index (%)	96.7	100.0	100.0	96.7	95.3 (80.0 – 100.0)
F0 Females
Estrous Cycle Length (days)	3.9 ± 0.39	4.0 ± 0.38	4.0 ± 0.23	4.0 ± 0.56	4.1 (3.9 – 4.4)
Precoital index (days)	2.1 ± 1.1	2.3 ± 0.93	2.4 ± 0.90	2.6 ± 2.40	2.8 (2.3 – 3.4)
Gestation length (days)	21.7 ± 0.45	21.6 ± 0.50	21.8 ± 0.50	21.4 ± 0.51	21.8 (21.6 – 22.1)
F0 Males
Sperm motility (%)	83	84	83	84	86 (81.0 – 92.0)
Spermatid count (M/g)	78.9 ± 22.1	77.6 ± 17.6	78.4 ± 22.9	75.6 ± 17.3	86.1 (69.2 – 111.8)
Cauda epididymis sperm count (M/g)	511.0 ± 149.8	528.7 ± 102.0	523.2 ± 99.90	522.8 ± 95.8	458.5 (381.8 – 624.7)
Sperm production rate (M/g/day)	12.9 ± 3.63	12.7 ± 2.89	12.8 ± 3.77	12.4 ± 2.84	14.1 (11.3 – 18.3)
Sperm Morphology (% Normal)	99.7	99.5	99.8	99.6	99.5 (97.7 – 99.9)

* Charles River Ashland historical control data (version 2021.01).

Mean values are presented ±standard deviation, where applicable (n = 25–30/sex/group).

None statistically significantly different from the control group.

 

Table 5. F1 generation - litter parameters and pup survival indices
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^aMean (Min - Max)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
Implantation Sites (#)	14.6 ± 2.3	14.9 ± 1.9	14.3 ± 2.5	15.2 ± 1.9	14.9 (14.2 – 16.2)
Pups Born (#)	13.9 ± 2.2	13.4 ± 1.9	13.4 ± 2.6	14.6 ± 1.9	13.9 (12.9 – 14.7)
Unaccounted Sites (#)	0.8 ± 0.7	0.7 ± 0.8	0.9 ± 1.3	0.6 ± 0.7	1.0 (0.7 – 1.6)
Live Litter Size (PND 0)	13.8 ± 2.1	14.1 ± 2.2	13.3 ± 2.6	14.5 ± 1.9	13.6 (12.5 – 14.4)
Males Per Litter (%)	48.6	48.1	45.5	45.3	50.2 (44.5 – 55.8)
Male Birth Weight (g, PND 1)	7.4 ± 0.57	7.4 ± 0.87	7.5 ± 0.87	7.0 ± 0.65	7.3 (6.8 – 7.7)
Female Birth Weight (g, PND 1)	7.0 ± 0.61	6.9 ± 0.82	7.1 ± 0.81	6.6 ± 0.65	6.8 (6.4 – 7.4)
Pup Survival (PND 0–4, % per litter)	98.8	94.8	94.7	93.5	93.2 (84.2 – 97.6)
Pup Survival (PND 4–21, % per litter)	99.0	89.3	99.7	96.2	97.5 (86.9 – 100)
Male Body Weight (g, PND 21)	50.9 ± 5.05	46.1 ± 6.66*	46.7 ± 7.15*	46.2 ± 4.61*	53.7 (42.5 – 62.8)
Female Body Weight (g, PND 21)	49.1 ± 4.41	43.9 ± 6.75**	45.2 ± 6.17*	44.6 ± 4.89*	51.6 (42.5 – 59.1)

^a Charles River Ashland historical control data (version 2021.01).

Mean values are presented ±standard deviation (n = 25–30/sex/group).

* Significantly different from control group at 0.05 using Dunnett’s test.

** Significantly different from control group at 0.01 using Dunnett’s test.

PND = Postnatal Day, # = Number.

 

Table 6. F1 generation – pre- and post-weaning developmental landmarks
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^aMean (Min - Max)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F1 Males
AGD - PND 1 (mm)	4.07 ± 0.234	4.09 ± 0.263	4.14 ± 0.223	4.07 ± 0.228	3.66 (2.0 – 4.1)
Adjusted AGD^b- PND 1	2.09 ± 0.105	2.11 ± 0.111	2.12 ± 0.108	2.13 ± 0.131	2.02 (1.6 – 2.2)
NR - PND 13	0.0	0.0	0.0	0.0	0.00 (0.0 – 0.0)
BPS – Age of Attainment (days)	44.3 ± 1.90	44.6 ± 1.90	44.2 ± 1.49	44.7 ± 1.99	44.3 (42.5 – 47.7)
BPS - BW at Attainment (g)	232 ± 22.6	230 ± 17.4	228 ± 16.1	230 ± 20.6	234.9 (210.3 – 255.1)
F1 Females
AGD - PND 1 (mm)	2.09 ± 1.31	2.15 ± 0.416	2.12 ± 0.203	2.14 ± 0.168	1.92 (1.0 – 2.3)
Adjusted AGD^b- PND 1	1.09 ± 0.069	1.13 ± 0.241	1.11 ± 0.103	1.14 ± 0.077	1.08 (0.9 – 1.2)
VP – Age of Attainment (days)	33.6 ± 1.46	34.9 ± 2.24*	33.5 ± 1.33	33.9 ± 1.56	33.5 (32.0 – 36.3)
VP - BW at Attainment (g)	119 ± 9.0	123 ± 14.3	116 ± 12.8	118 ± 9.2	121.2 (110.3 – 132.9)

^a Charles River Ashland historical control data (version 2021.01).

^b Adjusted AGD represents the anogenital distance relative to the cube root of body weight (no units).

Mean values are presented ±standard deviation, where applicable (n = 25–30/ sex/group).

* Significantly different from control group at 0.05 using Dunnett’s test.

AGD = Anogenital Distance; BPS = Balanopreputial Separation; BW = Body Weight; NR = Nipple (Areola) Retention; PND = Postnatal Day; VP = Vaginal Patency.

 

Table 7. F1 Generation (Cohort 1A) – Serum Total Thyroxine (T4, pg/mL) and Thyroid Stimulating Hormone (TSH, ng/mL) in Neonates (PND 4, pooled sexes) and in Weanling (PND 21) and Adult (PND 91) Male and Female Rats
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^a Mean (±2 SD)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F1 Litters (PND 4)
Total T4	15360 ± 2792	15352 ± 4594	15867 ± 3942	15229 ± 3690	22161 (15693 – 28630)
TSH	1.3 ± 1.38	0.6 ± 0.97	0.0 ± 0.0*	1.4 ± 1.08	4.37 (0.32 – 8.4)
F1 Males (PND 21)
Total T4	35240 ± 7728	36850 ± 12886	39970 ± 8914	45180 ± 9328	47030 (16477 – 77584)
TSH	3.9 ± 1.22	3.4 ± 1.72	2.2 ± 1.77	3.0 ± 1.94	4.91 (0.99 – 8.8)
F1 Females (PND 21)
Total T4	30280 ± 7638	39550 ± 6342*	38390 ± 7481	43030 ± 10125**	45236 (11484 – 78989)
TSH	2.6 ± 1.63	3.9 ± 1.85	2.3 ± 1.37	3.1 ± 2.01	4.62 (1.21 – 8.0)
F1 Males (Adult, PND 91)
Total T4	44950 ± 14463	54170 ± 8662	56670 ± 10481	56660 ± 7432	50791 (28279 – 73305)
TSH	16.6 ± 10.14	9.7 ± 4.13	11.6 ± 5.75	11.0 ± 5.82	8.41 (1.84 – 14.9)
F1 Females (Adult, PND 91)
Total T4	39740 ± 10356	42280 ± 42280	46700 ± 46700	43480 ± 43480	40920 (26819 – 55023)
TSH	7.6 ± 1.96	7.2 ± 2.75	7.0 ± 3.01	6.8 ± 3.54	5.21 (1.33 – 9.0)

^a Charles River Ashland historical control data (version 2020.02).

Mean values are presented ±standard deviation (n = 10/sex/group).

* Significantly different from control group at 0.05 using Dunnett’s test.

** Significantly different from control group at 0.01 using Dunnett’s test.

 

Table 8. F1 generation (cohort 1A and 1B) – reproductive performance and male sperm parameters
Dietary Concentration (ppm) Group	0	7500	11500	15000	HCD^a Mean (Min - Max)
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F1 Males and Females
Mating Index (%)	96.0	100.0	100.0	100.0	98.5 (93.3 – 100.0)
Fertility Index (%)	92.0	87.5	96.0	96.0	93.9 (80.0 – 100.0)
Copulation/Conception Index (%)	95.8	87.5	96.0	96.0	95.3 (80.0 – 100.0)
F1 Females
Primordial Follicle Counts^b	187.6 ± 64.5	-	-	249.3 ± 87.2	187.1 (110 – 323.7)
Age at First Estrus (Cohort 1A)	35.9 ± 2.9	36.7 ± 5.5	34.4 ± 2.3	35.9 ± 2.9	35.6 (33.7 – 38.3)
Estrous Cycle Length (days, Cohort 1A)	4.1 ± 0.15	4.2 ± 0.77	4.3 ± 0.72	4.3 ± 0.73	4.1 (3.9 – 4.4)
Estrous Cycle Length (days, Cohort 1B)	4.2 ± 0.64	4.4 ± 1.07	4.2 ± 0.50	4.4 ± 1.2	4.1 (3.9 – 4.4)
Precoital Index (days)	2.4 ± 1.21	2.7 ± 1.34	3.0 ± 1.10	2.7 ± 2.49	2.8 (2.3 – 3.4)
Gestation Length (days)	21.9 ± 0.63	21.9 ± 0.31	21.8 ± 0.42	21.8 ± 0.41	21.8 (21.6 – 22.1)
F1 Males
Sperm motility (%)	93	90	90	91	86 (81.0 – 92.0)
Spermatid count (M/g)	103.8 ± 24.2	104.9 ± 24.6	106.2 ± 30.1	102.6 ± 17.9	86.1 (69.2 – 111.8)
Cauda epididymis sperm count (M/g)	530.1 ± 133.8	420.5 ± 94.8**	457.1 ± 96.9*	438.9 ± 96.4**	458.5 (381.8 – 624.7)
Cauda Epididymis Weight (g)	0.2251 ± 0.055	0.2486 ± 0.027	0.2628** ± 0.025	0.2695** ± 0.030	0.2942 (0.2438 – 0.3675)
Sperm production rate (M/g/day)	17.0 ± 3.97	17.2 ± 4.03	17.4 ± 4.94	16.8 ± 2.93	14.1 (11.3 – 18.3)
Sperm Morphology (% Normal)	99.4	99.6	99.8	99.7	99.5 (97.7 – 99.9)

^a Charles River Ashland historical control data (version 2021.01).

^b Evaluated in the Control and 15,000 ppm groups only.

Mean values are presented ±standard deviation, where applicable (n = 20–25/sex/group).

* Significantly different from control group at 0.05 using Dunnett’s test.

** Significantly different from control group at 0.01 using Dunnett’s test.

Table 9. F1 generation (cohort 2A and 2B) – Brain weights and gross brain measurements
Dietary Concentration (ppm) Group	F1 Males					F1 Females
	0	7500	11500	15000	HCD^a Mean (Min-Max)	0	7500	11500	15000	HCD^a Mean (Min-Max)
Target Dose Level (mg/kg bw/day)	0	500	750	1000		0	500	750	1000
PND 22
Final Body Weight (g)	59 ± 9.3	53 ± 6.5	52 ± 8.6	48 ± 5.9**	55.9 (51.1 – 64.9)	51 ± 7.4	45 ± 11.7	49 ± 11.2	49 ± 7.8	52.7 (47.6 – 59.3)
Brain (g)	1.63 ± 0.08	1.60 ± 0.08	1.63 ± 0.06	1.61 ± 0.09	1.67 (1.61 – 1.73)	1.60 ± 0.10	1.52 ± 0.16	1.49 ± 0.12	1.53 ± 0.07	1.60 (1.53 – 1.66)
Brain Length (mm)	18.48 ± 0.38	18.28 ± 0.35	18.24 ± 0.49	18.20 ± 0.48	18.51 (18.1 – 18.8)	18.0 ± 0.59	17.8 ± 0.75	17.95 ± 0.49	17.87 ± 0.41	18.28 (17.9 – 18.7)
Brain Width (mm)	14.61 ± 0.45	14.69 ± 0.32	14.69 ± 0.37	14.62 ± 0.29	14.77 (14.4 – 15.0)	14.57 ± 0.28	14.46 ± 0.44	14.31 ± 0.49	14.51 ± 0.29	14.60 (14.4 – 14.9)
PND 78
Final Body Weight (g)	435 ± 30.7	431 ± 55.6	430 ± 39.7	444 ± 40.0	473 (457 – 486)	270 ± 20.9	258 ± 23.0	273 ± 17.1	261 ± 39.2	268 (249 – 287)
Brain (g)	2.33 ± 0.088	2.33 ± 0.176	2.33 ± 0.176	2.33 ± 0.092	2.38 (2.27 – 2.46)	2.15 ± 0.083	2.16 ± 0.107	2.17 ± 0.101	2.15 ± 0.093	2.16 (2.05 – 2.24)
Brain Length (mm)	21.72 ± 0.46	21.64 ± 0.62	21.73 ± 0.49	21.87 ± 0.46	21.83 (21.6 – 22.5)	21.09 ± 0.47	21.1 ± 0.45	21.12 ± 0.43	21.07 ± 0.51	20.94 (20.1 – 21.8)
Brain Width (mm)	15.59 ± 0.34	15.57 ± 0.41	15.59 ± 44	15.64 ± 0.30	15.68 (15.5 – 15.8)	15.31 ± 0.38	15.19 ± 0.20	15.22 ± 0.32	15.19 ± 0.35	15.21 (14.9 – 15.5)

^aCharles River Ashland historical control data (version 2021.01).

Mean values are presented ±standard deviation (n = 12/sex/group).

** Significantly different from control group at 0.01 using Dunnett’s test.

 

Table 10. F1 generation (cohort 2A) – mean brain morphometric measurements (1-Column)
	F1 Males^a			F1 Females^a
Dietary Concentration (ppm) Group	0	15000	HCD^bMean (Min-Max)	0	15000	HCD^bMean (Min-Max)
Target Dose Level (mg/kg bw/day)	0	1000		0	1000
Level 1
S1 - Thickness Frontal Cortex	1988 ± 95	1979 ± 78	2199 (2115 – 2304)	2053 ± 106	2002 ± 50	2155 (2024 – 2260)
S2 - Thickness Parietal Cortex	2419 ± 75	2443 ± 81	2389 (2302 – 2550)	2391 ± 86	2307 ± 93*	2243 (2054 – 2412)
S3 - Width Caudate-Putamen	4221 ± 133	4313 ± 97	4322 (4112 – 4581)	4179 ± 134	4173 ± 156	4146 (3923 – 4332)
S4 - Thickness Corpus Callosum	369 ± 35	372 ± 46	409 (355 – 536)	374 ± 33	362 ± 49	367 (325 – 445)
Level 2
S5 - Thickness Hippocampus	1646 ± 76	1684 ± 59	1701 (1657 – 1740)	1641 ± 64	1636 ± 47	1618 (1558 – 1709)
Level 3
S6 - Height Cerebellum	5965 ± 297	6016 ± 225	6147 (5618 – 6655)	5807 ± 199	5843 ± 297	5851 (5707 – 6098)

^a Evaluated in the Control and 15,000 ppm groups only.

^b Charles River Ashland historical control data (version 2021.01).

Mean values (μm) are presented ±standard deviation (n = 12/sex/group).

* Significantly different from control group at 0.05 using Dunnett’s test.

 

Table 11. F1 generation (cohort 1A) – splenic lymphocyte immunophenotyping – relative cell populations
Dietary Concentration (ppm) Group	0	7500	11500	15000
Target Dose Level (mg/kg bw/day)	0	500	750	1000
F1 Males
Total T lymphocytes (%)	41.3 ± 4.0	41.5 ± 7.3	42.3 ± 7.1	40.3 ± 6.7
Helper T lymphocytes (%)	26.6 ± 2.1	26.2 ± 5.6	26.8 ± 5.7	25.7 ± 3.6
Cytotoxic T lymphocytes (%)	13.9 ± 2.8	14.6 ± 3.3	14.7 ± 2.9	13.8 ± 4.3
B lymphocytes (%)	45.8 ± 3.3	46.9 ± 6.3	45.4 ± 6.0	47.3 ± 5.5
NK lymphocytes (%)	4.3 ± 1.9	3.8 ± 0.8	4.1 ± 1.5	4.0 ± 1.4
NK-T lymphocytes (%)	0.4 ± 0.13	0.4 ± 0.16	0.4 ± 0.17	0.4 ± 0.15
F1 Females
Total T lymphocytes (%)	44.4 ± 6.2	43.6 ± 6.1	41.8 ± 6.1	43.8 ± 6.1
Helper T lymphocytes (%)	28.1 ± 4.5	27.8 ± 4.0	26.4 ± 4.7	26.8 ± 4.7
Cytotoxic T lymphocytes (%)	15.4 ± 2.4	14.9 ± 2.8	14.3 ± 2.2	16.0 ± 2.6
B lymphocytes (%)	43.2 ± 5.5	43.3 ± 5.2	45.7 ± 5.9	44.0 ± 4.4
NK lymphocytes (%)	3.7 ± 0.8	3.8 ± 1.2	4.2 ± 1.0	3.8 ± 0.9
NK-T lymphocytes (%)	0.4 ± 0.07	0.4 ± 0.07	0.4 ± 0.13	0.5 ± 0.24

Mean values are presented ±standard deviation (n = 10/sex/group)

None significantly different from control group

Conclusions:

Based on the lack of any adverse treatment-related effects observed in the F0 (parental), F1 (first), and F2 (second) generation animals, the No Observed Adverse Effect level (NOAEL) for benzoic acid for systemic toxicity, reproductive toxicity, neonatal toxicity, developmental neurotoxicity, and immunotoxicity in the rat was determined to be 1000 mg/Kg bw/day.

Executive summary:

A key OECD Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study in rats was conducted to evaluate the effects that may occur as a result of pre- and post-natal exposure to the test material (Benzoic acid; CAS# 65-85-0). The test material was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats at concentrations of 0, 7,500, 11,500, and 15,000 ppm (equivalent to approximately 0, 500, 750, and 1000 mg/Kg bw/day, respectively). The study included reproductive assessments in the F1 generation and the F2 offspring, which were evaluated through postnatal day (PND) 91. In the F1 offspring, the study included evaluation of potential effects of the test material on reproduction, the developing neurological system, and the developing immune system, as well as learning and memory assessments.

 

Following a 2-week premating period, parental F0 generation animals were cohabitated overnight for mating (1 male with 1 non-sibling female of the same dose group) until positive evidence of mating (copulatory plug or sperm in a vaginal smear was determined (Gestation Day 0)) or three oestrous cycles had elapsed (a maximum of 14 days). F0 females were allowed to rear their litters normally until weaning on PND 21, subsequent to which randomly selected F1 pups were assigned to Cohorts 1, 2, and 3 such that not more than 1 pup/sex/litter was assigned to each cohort. F1 males and females assigned to Cohort 1B (optional reproductive assessments) were bred in a similar manner as the F0 adults, and females were allowed to rear their litters normally until weaning on PND 21, when pups/sex/litter were randomly selected to constitute the F2 generation.

 

All animals were observed twice daily for moribundity and mortality while clinical signs were monitored for once daily. Body weights were measured weekly throughout the study, and twice weekly during gestation and lactation for mated females. Food consumption was measured weekly throughout the study, and twice weekly during gestation and lactation for mated females. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported for each sex and group. Mean benzoic acid intake (on a mg/Kg bw/day basis) was calculated and reported for each sex and group. For the F0 and F1 generations, female oestrous cycles were monitored based on daily vaginal lavages (2 weeks prior to mating) and throughout the mating period, until evidence of mating (by the presence of a vaginal copulatory plug or sperm in a vaginal lavage). At termination, blood samples were collected from all parental males and females for the assessment of thyroid hormones and clinical pathology (haematology, serum chemistry and urinalysis). Following complete necropsy, tissues and organs were collected for organ weights and a full histopathological evaluation. Sperm parameters (motility, concentration and morphology) were evaluated for all males and a terminal vaginal lavage sample was collected for all females to determine the stage of the oestrous cycle at the time of necropsy.

 

F1 litters were observed for clinical signs and survival on PND 1, 4, 7, 14, and 21. Body weights were recorded and pups sexed on PND 0, 4, 14 and 21. The anogenital distance was measured for all pups on PND 1. To reduce variability among the litters, ten pups from each litter, of equal sex distribution (where possible) were randomly selected on PND 4. All male pups were examined for the presence of nipples/areolae on PND 13. All pups selected for Cohorts 1, 2, and 3 were examined for the attainment of postweaning developmental landmarks (vaginal patency [PND 25 onwards] and balanopreputial separation [PND 35 onwards]). Body weights were also recorded for individual pups on the day of attainment of the developmental landmarks. F1 animals designated for reproductive toxicity assessments were assigned to Cohorts 1A and 1B. In Cohort 1A, animals were evaluated for

onset of oestrous and oestrous cyclicity during the in-life period (PND 75–91), splenic lymphocyte immunophenotyping (PND 91) and thyroid hormones, sperm parameters, and clinical pathology, organ weights, and histopathology at termination (PND 91). F1 animals assigned to Cohort 1B were mated to produce an F2 generation to evaluate the effects of benzoic acid on F1 pup reproductive behaviour, parturition, litter parameters, offspring survival, growth and development until adulthood (PND 91).

 

F1 animals designated for developmental neurotoxicity assessments were assigned to Cohorts 2A and 2B. In Cohort 2A, animals were evaluated for neurobehavior (auditory startle response, functional observational battery (FOB), locomotor activity and learning and memory (Biel maze) assessment) during the in-life period, and at termination (PND 78), brain measurements (weight, length, width) and

neuropathology, including microscopic morphometric measurements on homologous stained sections of the brain.Animals assigned to Cohort 2B were used for brain measurements (weight, length, width) and neuropathological evaluation of brain on PND 22.

 

F1 animals designated for developmental immunotoxicity assessments were assigned to Cohort 3. Animals were evaluated for T-Cell Dependent Antibody Response (TDAR) at termination. In addition, 10 additional control rats/sex were assigned to Cohort 3A - a subset of Cohort 3 - which served as the positive control (cyclophosphamide) group.

 

Prior to PND 21, 2 F2 pups per sex from all available litters in Cohort 1B were selected to constitute the F2 generation. Following weaning, clinical signs, body weights and food consumption were measured weekly throughout the generation until termination (PND 91), at which all animals were subjected to a gross necropsy and brains were collected, weighed and preserved for histopathological evaluation.

 

F0 Generation

At all doses administered, benzoic acid had no effects on survival in the F0 generation and no benzoic acid-related clinical signs nor any significant effects on male or female body weights or food consumption. There was no effect of dietary benzoic acid administration on F0 reproductive performance, female oestrous cyclicity or sperm parameters. At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F0 males and females and there were no treatment-related gross macroscopic or microscopic observations at necropsy or any effects on clinical pathology (haematology, serum chemistry, urinalysis, and bile acids) or organ weights.

 

F1 Generation

Cohort 1: There were no benzoic acid-related clinical signs observed in the F1 generation through the study period and the mean number of pups born and post-natal survival in the F1 generation was unaffected by treatment. Pup body weights and body weight gains across all groups were comparable to the control group during PND 1–14. Although lower pup body weight gains were noted for both males and females during PND 14–21, the reductions in absolute mean body weights were within the range of the laboratory’s historical control data for reproduction studies and were non-dose responsive, therefore considered unrelated to benzoic acid administration. There were no significant effects on food consumption observed and haematological and clinical chemistry parameters were unaffected post exposure to the test material. Exposure to benzoic acid had no effect on pup pre-weaning or post-weaning developmental landmarks (anogenital distance, nipple retention) and gross necropsy did not reveal any remarkable findings. Organ weights of F1 animals were also unaffected by treatment and no treatment-related microscopic findings were observed during the histopathological evaluation.

 

At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F1 males and females. There was no effect of dietary benzoic acid administration on F1 reproductive performance, female oestrous cyclicity, time to first oestrus, female primordial follicle counts or sperm parameters. Higher mean cauda epididymal weights were observed in all treated groups when compared to the controls. Correspondingly, statistically significantly lower mean cauda epididymal sperm concentrations (millions/gram tissue) were noted in all benzoic acid-treated groups compared to the control group. However, these differences were not considered treatment-related but ascribed to the atypically low mean cauda epididymal weight and consequently higher mean cauda epididymal sperm concentration observed for the control group, which was attributed to 3 (of 20) males in the control group with atypically low values for cauda epididymal weight (0.0795–0.1502 g versus a historical control mean ± SD of 0.2942 ± 0.032 g).

 

Cohort 2: There were no treatment-related macroscopic findings or effects on gross brain measurements (weight, width, length) in F1 pups. There were also no treatment-related effects on microscopic morphometric measurements S1-S6 in the brains of young adult pups. In the neurobehavioral assessment, there were no effects of treatment on auditory startle responsiveness (peak response or time to peak response on PND 24), functional observational battery (FOB) and locomotor activity (ambulatory counts or total counts on PND 65) or learning and memory assessments on PND 66.

 

Cohort 3: There were no benzoic acid-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the TDAR Assay. The positive control (Cohort 3A), cyclophosphamide (CPS) produced the expected significant decrease in anti-sRBC-IgM in response to immunization with sRBC

 

F2 Generation

For pups selected to constitute the F2 generation, there were no benzoic acid related clinical signs nor any significant effects on male or female body weights or food consumption. Exposure to benzoic acid had no effect on absolute or relative (to final body weight) brain weights in any group of males or females in the F2 generation.

 

Based on the lack of any adverse treatment-related effects observed in the F0 (parental), F1 (first), and F2 (second) generation animals, the No Observed Adverse Effect level (NOAEL) for benzoic acid for systemic toxicity, reproductive toxicity, neonatal toxicity, developmental neurotoxicity, and immunotoxicity in the rat was determined to be 1000 mg/Kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimish 1 rating, study conducted in a GLP compliant lab according to current test Guidelines

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key screening reproductive/developmental toxicity data is available for propylene glycol dibenzoate (PGDB). This data is supported by relevant information available from a structural analogue Dipropylene glycol dibenzoate (DPGDB) and metabolite benzoic acid. 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 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.

 

Reproductive endpoints which were affected by oral gavage administration of PGDB included; an extended pre-coital interval for two animals and slightly lower mating evidence (sperm evidence in vaginal smears) for animals receiving 1000 mg/Kg bw/day. There was a slight decrease in live birth index and a small increase in the number of offspring dying between birth and Day 7 of age for the group receiving 1000 mg/Kg bw/day.

 

Male and female offspring body weight on Day 1 of age was low at 1000 mg/Kg bw/day; this was despite a tendency towards a longer gestation length which was probably secondary to restricted intra uterine growth. Offspring body weight gain thereafter remained slightly low at 300 or 1000 mg/Kg bw/day. Macroscopic examination at scheduled termination on Day 7 of age confirmed this with, a number of offspring in the 1000 mg/Kg bw/day group noted to be of thin build and with no milk in stomach. As the effects on offspring survival were limited to the 1000 mg/Kg bw/day group, the effects at 300 mg/Kg bw/day were considered not to be adverse at the degree observed.

 

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. The NOAEL for reproductive and developmental toxicity was also determined to be 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

In a key read across OECD Guideline 416 two generation study in rats (Huntingdon Life Sciences, 2001c; Klimisch score = 1) conducted to assess the effects of the test material DPGDB on reproductive performance, dietary administration of DPGDB at concentrations of 1000, 3300 or 10000 ppm was generally well tolerated by the P (F0) and subsequent F1 parental animals and their respective progeny. Exposure to the test material was in line with expectation throughout both generations fluctuations reflected the different physiological status of the animals and were predictably highest for females during peak lactation and in young animals. Bodyweight change of F1 females before paring and F1 males were slightly but significantly lower than in Controls.No adverse effects were seen on overall parental food consumption; food conversion efficiency calculated during the 10 week pre-mating phase was considered similar to controls for both generations.Oestrous cycle, mating performance, fertility and fecundity were similar in all groups. Gestation lengths and the parturition process were unaffected by treatment. Assessment of the terminal vaginal smears taken from F0 females revealed a higher incidence of females in oestrus in groups treated with DPGDB compared with controls. This finding was not apparent among F1 females and is considered to be of doubtful biological significance.

 

Litter parameters at birth of the F1 and F2 progeny and their survival to weaning showed no apparent detrimental effects of treatment with DPGDB. However, in both F1 and F2 offspring at 10000 ppm there was a slight reduction on weight gain during days 14-21 of age and this finding may be linked to the transition to direct exposure to the test material as the offspring weaned on to solid diet at the same dietary inclusion levels as their parents.

 

No treatment related findings were seen at microscopic examination of the F1 offspring not selected to form the next generation or the F2 offspring killed after weaning.Macropathology, histopathology assessment and sperm analysis for the F0 and F1 adults showed no adverse effects of treatment.

 

The only possible effect of treatment detected at assessment of organ weights from F1 and F2 offspring was significantly lower absolute and relative spleen weight among F2 males and females compared to controls. The toxicological significance if this finding is uncertain since it was not detected among F1 offspring or among F0/F1 adult animals.The evidence from this study suggested that a dietary concentration of DPGDB at 10000 ppm should be considered as the No Observed Adverse Effect Level (NOAEL) for P (F0) and F1 parent animals. The NOAEL for developing offspring is considered to be 3300 ppm. The No Observed Effect Level (NOEL) for reproductive parameters is considered to be 10000 ppm.

The evidence from this study suggested that a dietary concentration of DPGDB at 10000 ppm should be considered as the No-Observed-Effect-Level (NOEL) for F0 and F1 parent animals. The No-Observed-Adverse-Effect-Level (NOAEL) for survival and growth of the offspring is considered to be 10000 ppm (equivalent to a minimum estimated daily achieved dosage of 500 mg/Kg bw/d).

An OECD Guideline 443 Extended One-Generation Reproductive Toxicity Study in Sprague Dawley rats (Turnbull et al, 2021; Klimisch score = 1) at concentrations of 0, 7,500, 11,500, and 15,000 ppm (equivalent to approximately 0, 500, 750, and 1000 mg/kg bw/day, respectively) was conducted to assess the effects of the test material benzoic acid, the main metabolite of PGDB. The study included reproductive assessments in the F1 generation and the F2 offspring, which were evaluated through postnatal day (PND) 91. In the F1 offspring, the study included evaluation of potential effects of the test material on reproduction, the developing neurological system, and the developing immune system, as well as learning and memory assessments (cohorts 1A, 1B, 2A, 2B and 3).

 

F0 Generation

At all doses administered, benzoic acid had no effects on survival in the F0 generation and no benzoic acid-related clinical signs nor any significant effects on male or female body weights or food consumption. There was no effect of dietary benzoic acid administration on F0 reproductive performance, female oestrous cyclicity or sperm parameters. At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F0 males and females and there were no treatment-related gross macroscopic or microscopic observations at necropsy or any effects on clinical pathology (haematology, serum chemistry, urinalysis, and bile acids) or organ weights.

 

F1 Generation

Cohort 1: There were no benzoic acid-related clinical signs observed in the F1 generation through the study period and the mean number of pups born and post-natal survival in the F1 generation was unaffected by treatment. Pup body weights and body weight gains across all groups were comparable to the control group during PND 1–14. Although lower pup body weight gains were noted for both males and females during PND 14–21, the reductions in absolute mean body weights were within the range of the laboratory’s historical control data for reproduction studies and were non-dose responsive, therefore considered unrelated to benzoic acid administration. There were no significant effects on food consumption observed and haematological and clinical chemistry parameters were unaffected post exposure to the test material. Exposure to benzoic acid had no effect on pup pre-weaning or post-weaning developmental landmarks (anogenital distance, nipple retention) and gross necropsy did not reveal any remarkable findings. Organ weights of F1 animals were also unaffected by treatment and no treatment-related microscopic findings were observed during the histopathological evaluation.

 

At termination, thyroid hormone (T4 and TSH) levels were unaffected by benzoic acid intake in F1 males and females. There was no effect of dietary benzoic acid administration on F1 reproductive performance, female oestrous cyclicity, time to first oestrus, female primordial follicle counts or sperm parameters. Higher mean cauda epididymal weights were observed in all treated groups when compared to the controls. Correspondingly, statistically significantly lower mean cauda epididymal sperm concentrations (millions/gram tissue) were noted in all benzoic acid-treated groups compared to the control group. However, these differences were not considered treatment-related but ascribed to the atypically low mean cauda epididymal weight and consequently higher mean cauda epididymal sperm concentration observed for the control group, which was attributed to 3 (of 20) males in the control group with atypically low values for cauda epididymal weight (0.0795–0.1502 g versus a historical control mean ± SD of 0.2942 ± 0.032 g).

 

Cohort 2: There were no treatment-related macroscopic findings or effects on gross brain measurements (weight, width, length) in F1 pups. There were also no treatment-related effects on microscopic morphometric measurements S1-S6 in the brains of young adult pups. In the neurobehavioral assessment, there were no effects of treatment on auditory startle responsiveness (peak response or time to peak response on PND 24), functional observational battery (FOB) and locomotor activity (ambulatory counts or total counts on PND 65) or learning and memory assessments on PND 66.

 

Cohort 3: There were no benzoic acid-related effects on the humoral immune system as indicated by lack of any changes in the sRBC-IgM responses in the TDAR Assay. The positive control (Cohort 3A), cyclophosphamide (CPS) produced the expected significant decrease in anti-sRBC-IgM in response to immunization with sRBC

 

F2 Generation

For pups selected to constitute the F2 generation, there were no benzoic acid related clinical signs nor any significant effects on male or female body weights or food consumption. Exposure to benzoic acid had no effect on absolute or relative (to final body weight) brain weights in any group of males or females in the F2 generation.

 

Based on the lack of any adverse treatment-related effects observed in the F0 (parental), F1 (first), and F2 (second) generation animals, the No Observed Adverse Effect level (NOAEL) for benzoic acid for systemic toxicity, reproductive toxicity, neonatal toxicity, developmental neurotoxicity, and immunotoxicity in the rat was determined to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Only screening reproductive/developmental toxicity 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.

PGDB

OECD 422 (Rat): Developmental Toxicity NOAEL = 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

DPGDB

OECD 414 (Rat): Developmental Toxicity NOAEL = 500 mg/Kg bw/day, based on increase in the number of fetuses with cervical ribs at 1000 mg/Kg bw/day.

OECD 414 (Rabbit): Developmental Toxicity NOAEL = 250 mg/Kg bw/day, based on lower mean fetal weights at 500 mg/Kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-25 to 2018-01-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company (United States); Batch no. V988701802
- Expiration date of the lot/batch: None
- Purity test date: 2017-01-25

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (18°C to 24°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: previously shown to be stable over the range of concentrations used on this studyfor at least 10 days under refrigerated (2°C to 8°C) conditions

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

OTHER SPECIFICS:
Purity: > 99.0%

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hra:(NZW)SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Covance Research Products, Inc. (Denver, PA)
- Age at study initiation: approximately 7 months old at the time of mating by the supplier
- Weight at study initiation: 2949 g to 3901 g on Gestation Day 0
- Fasting period before study: No
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel-O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding was changed at least twice weekly. Nesting material was not required because the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rabbit LabDiet®5322 - The basal diet was offered in 25-g increments 3 times per dayon the dayof arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the dose administration period; basal diet was offered ad libitum thereafter. Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study. Kale present in the cage at the time of providing a new leaf was discarded. The diet was supplemented with extra kale, celery sticks, and haycubes as necessary.
- Water (e.g. ad libitum): Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, was provided ad libitum
- Acclimation period: time-mated rabbits received on Gestation Day 1, 2, 3, or 4.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 61°F to 71°F (16°C to 22°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours) / 12-hour dark photoperiod

IN-LIFE DATES: From: 2017-06-30 To: 2017-07-28

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

medium viscosity, 400–800 cps, USP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Following administration of each dose, the catheter was flushed with 5 mL of deionized water to ensure delivery of the entire dose.

VEHICLE
- Justification for use and choice of vehicle (if other than water): carboxymethylcellulose (CMC); reason for choice not specified
- Concentration in vehicle: 0, 20, 50, or 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): Lot Nos. 2GA0044 and 1GE0858, retest dates: 31 Jul 2017 and 16 Apr 2018, respectively
- Purity: 0.5% carboxymethylcellulose in deionized water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A validated HPLC/UV method was used and extended for the determination of DPGDB concentration in suspension formulations. Method specificity/selectivity, calibration reproducibility, precision, and accuracy and were assessed and validated, satisfying SOP specified criteria. In addition, the results of the assessment of test substance homogeneity and, following 10 days of refrigerated storage, resuspension homogeneity in formulations prepared at target concentrations of 20 and 100 mg DPGDB/mL met the applicable protocol-specified acceptance criteria.

Details on mating procedure:

One hundred six time-mated female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Denver, PA, on 30 Jun 2017. The time-mated rabbits were received on Gestation Day 1, 2, 3, or 4.

Duration of treatment / exposure:

The vehicle and test substance formulations were administered orally by gavage, via appropriately sized rubber catheters, once daily during Gestation Days 7–28. The dose volume for all groups was 5 mL/kg. Dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study.

Frequency of treatment:

once daily during Gestation Days 7–28

Duration of test:

during Gestation Days 7–28.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control (Group 1)

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

500 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 24 rabbits/group.


Group Number Treatment Dosage/Dose Level Number of
Females
(mg/kg/day)

1 Vehicle Control 0 24
2 DPGDB 100 24
3 DPGDB 250 24
4 DPGDB 500 24

Control animals:

yes

Details on study design:

- Dose selection rationale:
Dosage levels were selected based on results of a previous dose range-finding prenatal developmental toxicity study in rabbits with DPGDB, and were provided by the Sponsor
Representative after consultation with the Charles River Study Director. In the previous study, time-mated rabbits were dosed (via oral gavage) with 100, 250, 500, and 1000 mg/kg/day of DPGDB during Gestation Days 7–28. Significant toxicity was noted at the 1000 mg/kg/day dosage level as evidenced by abortions, body weight losses, and reduced food consumption, resulting in early termination of this dosage group. At the 500 mg/kg/day dosage level, excreta-related findings were noted without significant effects on body weights or food consumption. Dosage levels of 100 and 250 mg/kg/day were well tolerated. Therefore, dosage levels of 100, 250, and 500 mg/kg/day were selected for the current study to provide the evaluation of potential dose-response and determination of a no-observed-adverse-effect level (NOAEL).

The selected route of administration for this studywas oral (gavage) because this is a potential route of exposure for humans.

- Rationale for animal assignment (if not random): The bred females were assigned to groups using a WTDMS™ computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations were recorded daily from the dayof receipt through Gestation Day 29 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1, 2, and 4 hours following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on Gestation Days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 5, and 7–29. Group mean body weights were calculated for each of these days. Mean bodyweight changes were calculated for each corresponding interval and also for Gestation Days 7–10, 10–13, 13–20, 20–29, and 7–29. Gravid uterine weight was collected and net bodyweight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Individual food consumption was recorded on Gestation Days 5–29. Food intake was reported as g/animal/dayand g/kg/dayfor the corresponding bodyweight change intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: Gestation Day 29 Laparohysterectomy: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovarywas recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. Maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. In addition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.

Unscheduled Deaths: For females that were found dead, euthanized in extremis, or aborted during the course of the study, maternal tissues with gross lesions were preserved in 10% neutral-buffered formalin for possible future histopathologic examination. Inaddition, the adrenal glands, stomach, heart, and kidneys were preserved in 10% neutral-buffered formalin for microscopic examination.The number and location of implantation sites, corpora lutea, and viable fetuses were recorded.

OTHER:

CLINICAL PATHOLOGY:
Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 females/group from all groups at necropsy (Gestation Day29), and from females that were euthanized in extremis or exhibited signs of toxicity during the study. For females euthanized in extremis, blood was collected prior to euthanasia, and animals were euthanized immediatelyfollowing blood collection by an intravenous injection of sodium pentobarbital via a marginal ear vein. The animals were not fasted prior to blood collection. Blood samples were collected from a marginal ear vein (or other site as deemed necessary). Blood was collected in tubes containing K2EDTA (hematology) or no anticoagulant (serum chemistry).

Parameters listed in Table 2. and Table 3. were evaluated for Haematology and Clinical Chemistry, respectively.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. Selected tissues were examined microscopically from all females found dead and euthanized in extremis, as well as from 5 females/group at the scheduled necropsy.

Fetal examinations:

The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations

- External examinations: Yes: all per litter
The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. Crown-rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.

- Soft tissue examinations: Yes: all per litter
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development.

- Skeletal examinations: Yes: all per litter
Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations

- Head examinations: Yes: all per litter. Heads from all fetuses were examined bya midcoronal slice.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Where applicable, the litter was used as the experimental unit.

Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA9 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test10 was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.

Indices:

Intrauterine data were summarized using 2 methods of calculation.

1) Group Mean Litter Basis:

Post implantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group / No. Gravid Females/Group)

2) Proportional Litter Basis:

Summation Per Group (%) = (Sum of Post implantation Loss/Litter (%) / No. Litters/Group)

Where:
Post implantation Loss/Litter (%) = No. Dead Fetuses, Resorptions (Early/Late)/Litter / No. Implantation Sites/Litter) x 100

Fetal developmental findings were summarized by:
1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and
2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:

Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%) / No. Litters/Group)

Where:
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter / No. Viable Fetuses/Litter) x 100

Historical control data:

Charles River Ashland historical control data was used (Appendix 6 of the study report)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Female in the 500 mg/kg/day group showed incidences of decreased defecation at the daily examinations during Gestation Days 20–26. Additional adverse clinical observations included rales, labored respiration, decreased respiration rate, and a thin body were noted at the daily examinations and/or postdosing observations during Gestation Days 23–26.

In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 with clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.In the 100 mg/kg/day group, Female was euthanized with clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related.

Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Test substance-related effects on survival were noted in the 500 mg/kg/day group.

One female in the 500 mg/kg/day group was euthanized in extremis on Gestation Day 26 following a severe body weight loss (18.3% during Gestation Days18–26) and markedly reduced food consumption (0 to 10 g/day during Gestation Days 19–26), with corresponding incidences of decreased defecation at the daily examinations during Gestation Days 20–26. In addition, Female in the 500 mg/kg/day group aborted 1 late resorption (with no apparent malformations) on Gestation Day 25, and was subsequently euthanized; however, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. At necropsy, neither female was noted with remarkable macroscopic findings. Due to the adverse clinical observations, body weight loss, reduced food consumption, and no clear cause of moribundity or abortion at necropsy, these findings were considered test substance-related and adverse.

No other test substance-related effects on survival were noted during the study. In the 500 mg/kg/day group, Female was found dead on Gestation Day 26 following a severe body weight loss (16.0% during Gestation Days 20–25) and markedly reduced food consumption (0 to 7 g/day during Gestation Days 21–25), and clinical observations of rales, labored respiration, and red material around the nose at the daily examinations and/or postdosing observations on Gestation Day 25. At necropsy, Female No. 2474 had dark red discoloration and firmness of all lobes of the left lung, which correlated microscopically with acute inflammation. The trachea was also noted to be filled with dark red contents, which microscopically was characterized by hemorrhage, edema, and dilatation of the submucosa. The cause of death of this female was considered acute inflammation of the lungs, most likely due to an intubation error, and not considered test substance-related.


In the 100 mg/kg/day group, Female was euthanized in extremis on Gestation Day 25 following a body weight loss (13.6% during Gestation Days 14–25), reduced food consumption (0 to 44 g/day during Gestation Days 16–25), and corresponding incidences of decreased defecation at the daily examinations during Gestation Days 19–20. Additional clinical observations of rales, labored respiration, a thin body, and/or clear discharge from the eyes and nose were noted for this female at the daily examinations and/or postdosing observations during Gestation Days 24–25. At necropsy, mottling and failure to fully inflate were observed in all lobes of the lungs, which correlated with acute inflammation microscopically. Acute inflammation was centered on the bronchi and bronchioles and was consistent with an intubation error; therefore, the cause of moribundity was acute inflammation of the lungs, and not considered test substance-related. In addition, Female in the 100 mg/kg/day group was found dead on Gestation Day 13. No remarkable effects on body weight or food consumption were noted prior to death, but a clinical observation of rales was noted for this female at the daily examinations and postdosing observations up to 2 days prior to death. At necropsy, oily contents in all lung lobes and a perforation of the trachea at approximately the level of cervical vertebra No. 3 were noted. Microscopically, the lungs showed acute inflammation. Perforation of the trachea was not confirmed histologically, but findings of edema, hemorrhage, dilatation, and mixed inflammatory cell infiltrates in the tracheal submucosa were likely related. Therefore, the cause of death for this female was acute inflammation of the lungs due to an intubation error, which was not considered test substance-related. Female in the 100 mg/kg/day group was euthanized in extremis on Gestation Day 24; no clinical observations or noteworthy changes in body weight or food consumption were noted prior to euthanasia. At necropsy, oily contents in all lung lobes and multiple, dark red, irregularly shaped areas in all lobes were noted for this female. Microscopically, the lungs showed acute inflammation and hemorrhage. Intubation error was determined to be the cause of debility and euthanasia, and therefore was not considered test substance-related. All other females survived to the scheduled euthanasia.


Test substance-related incidences of decreased defecation was noted for 4 surviving females in the 500 mg/kg/day group at the daily examinations during Gestation Days 15–29, which corresponded to and were considered secondary to the reduced food consumption noted in this group. There were no other test substance-related clinical observations noted at the daily examinations or approximately 1, 2, or 4 hours following dose administration. Observations
noted in the treated groups, including rales, labored respiration, and/or decreased respiration rate, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group.

Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group compared to the control group beginning on Gestation Day 13, and continuing sporadically throughout the remainder of the treatment period, resulting in lower mean body weight gains in this group when the Gestation Days 13–20 and 20–29 cumulative intervals and when the entire treatment period (Gestation Days 7–29) were evaluated. Although
none of the differences were statistically significant when compared to the control group and were not of sufficient magnitude to affect absolute mean body weights in this group, these changes were considered adverse as they led to the euthanasia or abortion of 2 females within the group. A mean net body weight loss (not statistically significant) was noted in the 500 mg/kg/day group compared to the control group, while mean net body weight and gravid
uterine weight in this group were comparable to the control group. Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 100 and 250 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significant (p < 0.05) mean body weight losses were noted in the 100 mg/kg/day group compared to the control group during Gestation Days 15–16 and 20–21; however, these transient differences did not occur in a dose-related manner, and were therefore not considered test substance-related. Mean net body weight losses were also noted in the 100 and 250 mg/kg/day groups; however, the differences were not statistically significant and did not occur in a dose-related manner, and were therefore not considered test substance-related.

Mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were
comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group.

Mean maternal food consumption, evaluated as g/animal/day or g/kg/day, in the 500 mg/kg/day group was comparable to the control group during the first week of treatment (Gestation Days 7–10 and 10–13), but generally lower than the control group during the remainder of the treatment period (Gestation Days 13–20 and 20–29); differences were significant (p < 0.05 or p < 0.01) during Gestation Days 21–23 and 24–25. The lower mean food consumption corresponded to the lower mean body weight gains noted in this group during the same period, and resulted in a slightly lower mean food consumption when the entire treatment period (Gestation Days 7–29) was evaluated, and in the presence of other adverse effects noted at this dosage level, was considered test substance-related and adverse.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in hematology parameters on Gestation Day 29. Increases in the percentage and the absolute number of monocytes were noted at 500 mg/kg/day compared to the control group. However, the differences were not statistically significant and could be attributed to a single female in this group, and in the absence of any other effects on hematology parameters, were considered to be the result of normal biological variation. There were no other effects on hematology parameters; there was no dose-response relationship, the changes were of minimal magnitude, and were not considered to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

There were no test substance-related changes in serum chemistry parameters on Gestation Day 29. There was no dose-response and the changes were of minimal magnitude. These differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered nonadverse. There were no other test-substance-related microscopic observations at any dosage level.

Adrenal cortical hypertrophy was considered a nonadverse finding due to the minimal to mild severity and the lack of other related microscopic and gross findings. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There were no test substance-related alterations in the prevalence, severity, or histologic character of those incidental tissue alterations.

Dosage (mg/kg/day): Females
0 100 250 500
Adrenal cortex 5 5 5 5
Hypertrophy 0 1 3 3
Minimal - 0 1 1
Mild - 1 2 2

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day group, 1 (4.2%) abortion was observed. However, no clinical observations or remarkable effects on body weights and food consumption were noted for this female prior to aborting. No apparent malformations and in female no remarkable macroscopic findings were observed. There were total 22 (91.7%), 23 (95.8%), 23 (95.8%), and 23 (95.8%) gravid females at the time of scheduled necropsy in Group 1, 2, 3, and 4 respectively (See Table 4).

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Preimplantation loss was observed in 19 fetus in group 1, 13 fetus in group 2, 20 fetus in group 3 and 14 fetus in group 4.Post-implantation loss was observed in 10 fetus in group 1, 6 fetus in group 2, 11 fetus in group 3 and 6 fetus in group 4. The mean percentages for Preimplantation loss were 8.3,6.5, 8.6 and 6.2% in Group 1,2,3 and 4. The mean percentages for Post implantation loss were 5.8,3.2, 4.3 and 3.1% in Group 1,2,3 and 4. Differences from the control group were not statistically significant.

Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. Non significant changes were observed (Refer attached tables) .

Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

Total 32 resorptions were observed in out of total 760 fetus. Total 10 resorptions in Group 1, and 6 resorptions in Group 2 and 10 resorptions in Group 3 and 6 resorptions in Group 4 were observed. The mean percentages for total resorption were 5.8, 3.2, 3.9 and 3.1% in Group 1, 2, 3, and 4 where Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and Group 4 is 500 mg/kg/d. The changes were non significant in comparisn to controls. Differences from the control group were slight and not statistically significant (See Tables 4 and 7) .

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Early resorption was observed in 9 fetus in group 1, 6 fetus in group 2, 2 fetus in group 3 and 3 fetus in group 4. Late resorption was observed in 1 fetus in group 1, 0 fetus in group 2, 8 fetus in group 3 and 3 fetus in group 4. The mean percentages for early resorption were 0.5, 0.0, 3.0 and 1.5% in Group 1,2,3 and 4. Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d. The changes were non significant in comparison to controls.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

1 fetus was found dead in group 3 (250 mg/kg/d). There were total 88 male viable fetus and 117 female viable fetus. out of total 205 viable fetus one dead fetus was found and which was non dose related. The mean percentages were 0.0,0.0,0.4 and 0.0 % for group 1,2,3 and 4. The values were not significantly different from control group.Group 1 is 0 mg/kg/d and Group 2 is 100 mg/kg/d group 3- 250 mg/kg/d and group 4 is 500 mg/kg/d.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Total 22 (100 %), 23 (91.7%) 23 (91.7%) and 23 (91.7%) number of females were gravid in Group 1,2,3 and 4. The non gravid preganicies were 2 (8.3%), 1 (4.8%), 1 (4.2%), 1 (4.8%) in Group 1,2,3 and 4.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant (Attached summary table below).

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Abnormalities:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean male, female, and combined fetal weights in the 500 mg/kg/day group were 8.9%, 11.8%, and 10.5% lower, respectively, compared to the control group, which corresponded with maternal toxicity observed at this dosage level. The differences were generally significant (p < 0.05 or p < 0.01) and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group.

The fetal weight in group 1 was 42.1 in grams and in group 2 was 42.0 gm and group 3 was 41.4 gm and in group three was 37.7 gm in 500 mg/kg/d. The group 4 was statistically significant different from the control group (See Table 7).

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Viable fetus in group 1 were 192 and group 2 were 178 and in group 3 were 205 and group 4 were 185. The results were comparable to controls and were non significant. The viable percentages values were 94.2, 96.8, 95.7 and 96.9 in Group 1, 2 , 3, and 4. Group 1 is 0 mg/kg/d; Group 2 is 100 mg/kg/d; Group 3 is 250 mg/kg/d; and Group 4 is 500 mg/kg/d. The mean values for vaible fetus in group 1, 2, 3, and 4 were 8.7 (0 mg/kg/d) , 8.9 (100 mg/kg/d), 8.9 (250 mg/kg/d) and 9.3 (500 mg/kg/d). The values showed that there were no treatment related effcts and values were comparable to controls.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was similar across all groups

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations were observed in fetuses (litters) in dose groups and were considered spontaneous in origin.

No test substance-related external malformations were noted for fetuses at any dosage level. In the 500 mg/kg/day group, one fetus was noted with a short tail (approximately 7 mm in length), one fetus was noted with exencephaly with open eyelids, and other fetus in same group was noted with a malpositioned umbilicus (located more lateral than normal). Skeletally, the short tail consisted of fused, malpositioned, and absent caudal vertebrae, and exencephaly consisted of small and misshapen frontal and parietal bones, an absent interparietal bone, and an unossified supraoccipital bone. These findings were noted in single fetuses, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data; therefore, they were not considered test substance-related. No other external malformations were noted in the test substance-treated groups. In the control group, fetus noted with carpal flexure (right) and polydactyly (6 digits present on the right forepaw), and fetus was noted with macroglossia. Skeletally, polydactyly consisted of one extra digit with only the distal phalanx ossified. There were no other external malformations observed on this study. No external developmental variations were noted in the test substance-treated groups. The only finding observed (excessive fat pads) was noted in a single fetus in the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.

Severely malaligned sternebrae, resulting in fused or attached sternebrae, were noted for 1 and 3 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition, sternoschisis (sternal band No. 2 not joined) and a vertebral centra anomaly (fused sacral centra) were noted for two Fetus in the 500 mg/kg/day group, respectively. Furthermore, one Fetus in the 250 mg/kg/day group was noted with a rib anomaly (left rib Nos. 3 and 4 were fused proximally through medially). A vertebral anomaly with or without associated rib anomaly, consisting of extra ribs and centra, malpositioned centra and arches, fused centra, arches, and ribs, bipartite centra, absent centra and arches, and malproportioned arches and centra, was noted for 1 and 2 fetuses in the 100 and 500 mg/kg/day groups, respectively. In addition costal cartilage anomalies were noted for three Fetus in the control, 100, 250, and 500 mg/kg/day groups, respectively; 2 of these fetuses were also noted with other skeletal malformations, as noted above. Because the aforementioned malformations were noted infrequently or at a similar frequency in the control group, were not observed in a clear dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data, they were not considered test substance-related. There were no other skeletal malformations observed on this study.


A higher mean litter proportion of the skeletal developmental variation pubis unossified was noted in the 500 mg/kg/day group (1.4% per litter) compared to the control group (0.0% per litter). The value in this group also exceeded the maximum mean value in the Charles River Ashland historical control data (0.58% per litter). This reduction in ossification was considered secondary to the lower mean fetal body weights noted at this dosage level . Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related visceral malformations were noted for fetuses at any dosage level.

Lobular agenesis of the lungs (absent right accessory lobe) was noted for 2 fetuses in the 250 mg/kg/day group. In the 100 mg/kg/day group, a bulbous aortic arch, vestigial pulmonary trunk, and interventricular defect (a 1 mm in diameter opening in the anterior portion of the septum) were noted for one fetus . Because these visceral malformations were noted in single fetuses, in the control group at similar or higher frequencies (in five fetus), and/or were not observed in the high-dose group, they were not considered test substance-related. No other visceral malformations were noted in the test substance-treated groups. In the control group, one fetus was also noted with a three chambered heart (2 atria and 1 ventricle with an absent tricuspid valve) and one fetus was also noted with a small right ventricle in the heart. There were no other visceral malformations observed on this study.

There were no test substance-related developmental variations noted at any dose level. A higher mean litter proportion of the visceral developmental variation pale spleen was noted in the 500 mg/kg/day group (3.1% per litter) compared to the control group (0.7% per litter). The value in this group exceeded the maximum mean value in the Charles River Ashland historical control data (1.77% per litter). However, because this finding was only noted for 6(3) fetuses (litters), and was not statistically significantly different from the control group, it was not considered test substance-related. Other findings observed in the test substance-treated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River
Ashland historical control data.

A cystic oviduct was noted for 1 and 2 fetuses in the 100 and 250 mg/kg/day groups, respectively, renal papilla(e) not fully developed (Woo and Hoar Grade 1) was noted for 1 and 2 fetuses in the 250 and 500 mg/kg/day groups, respectively, and red fluid in the abdominal cavity was noted for 1 fetus in the 500 mg/kg/day group. These findings were not classified as either a malformation or developmental variation and were not considered to be test substance-related because they occurred infrequently and/or in a manner that was not dose-related.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Table 4. Summary of Maternal Survival and Pregnancy Status
Dose Group (mg/Kg/day)	0 (Control)		100		250		500
	No.	%	No.	%	No.	%	No.	%
Females on Study	24		24		24		24
Females that aborted or delivered	0	0.0	0	0.0	0	0.0	1	4.2
Females that Died	0	0.0	1	4.2	0	0.0	1	4.2
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Non Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	1	100.0	0	0.0	1	100.0
Females that were euthanized	0	0.0	2	8.3	0	0.0	1	4.2
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Non Gravid	0	0.0	2	100.0	0	0.0	1	100.0
Females examined at Scheduled Necropsy	24	100.0	21	87.5	24	100.0	21	87.5
Non Gravid	2	8.3	1	4.8	1	4.2	1	4.8
Gravid	22	91.7	20	95.2	23	95.8	20	95.2
With resorptions only	0	0.0	0	0.0	0	0.0	0	0.0
With viable foetuses	22	100.0	20	100.0	23	100.0	20	100.0
Total Females Gravid	22	91.7	23	95.8	23	95.8	23	95.8

Table 5. Summary of Clinical Findings: Total Occurrence / No. of Animals
	Dose Group (mg/Kg/day)
	0 (Control)	100	250	500
Normal – No significant clinical observations	646/24	623/24	652/24	611/24
Disposition
- Found dead	0/0	1/1	0/0	1/1
- Sent to necropsy in extremis	0/0	2/2	0/0	1/1
- Aborted	0/0	0/0	0/0	1/1
- Scheduled Euthanasia; Gestation Day 29	24/24	21/21	24/24	21/21
Body/Integument
- Hair loss forelimb (s)	0/0	2/1	0/0	6/2
- Thin	0/0	1/1	0/0	2/2
- Hair loss Hindlimb (s)	0/0	0/0	0/0	1/1
Cardio-Pulmonary
- Rales	3/1	4/3	3/2	9/3
- Increases Respiration Rate	0/0	0/0	1/1	0/0
- Laboured Respiration	0/0	2/1	1/1	4/2
- Pale Body	0/0	0/0	1/1	0/0
Eyes / Ears / Nose
- Clear Discharge Left Eye	0/0	2/1	0/0	0/0
- Clear Discharge Right Eye	0/0	2/1	0/0	0/0
- Dried Red Material Around Nose	0/0	0/0	0/0	1/1
- Clear Nasal Discharge	0/0	1/1	0/0	0/0
Excreta – Decreased Defaecation	0/0	3/2	1/1	21/5
Body / Integ II
- Scabbing Dorsal Trunk	3/1	0/0	0/0	0/0
- Wet Brown Material Anogenital Area	1/1	0/0	0/0	0/0
- Dried Brown Material Anogenital Area	3/3	0/0	0/0	2/2

Table 6. Summary of Body Weight Changes during Gestation (g)
Parameter		Group (mg/Kg/day)
		0 (control)	100	250	500
Initial Body Weight	Mean	3314.	3357.	3321.	3348.
	S.D.	160.7	232.7	192.1	205.2
	S.E.	34.2	52.0	40.1	45.9
	N	22	20	23	20
Terminal Body Weight	Mean	3826.	3816.	3811.	3735.
	S.D.	229.0	236.2	254.4	344.9
	S.E.	48.8	52.8	53.1	77.1
	N	22	20	23	20
Gravid Uterine Weight	Mean	497.9	510.0	516.2	487.4
	S.D.	113.98	79.44	98.45	87.61
	S.E.	24.30	17.76	20.53	19.59
	N	22	20	23	20
Net Body Weight	Mean	3328.3	3306.1	3295.2	3247.1
	S.D.	235.97	222.21	235.63	285.10
	S.E.	50.31	49.69	49.13	63.75
	N	22	20	23	20
Net Body Weight Change	Mean	14.7	-51.3	-25.3	-100.5
	S.D.	191.80	191.90	146.80	275.20
	S.E.	40.89	42.91	30.61	61.54
	N	22	20	23	20

Mean Differences Calculated from Individual Differences

Non Gravid Weight(s) Not Included in Calculation of Mean

Table 7. Summary of Fetal Data at Scheduled Necropsy
Group (mg/Kg/day)		Sex		Viable Fetuses	Dead Fetuses	Resorptions		Post Implantation Loss	Implantation Sites	Corpora Lutea	Pre-Implantation Loss	Fetal Weight in Grams	No. of Gravid Females
		Male	Female			Early	Late
0 (Control)	Total	92	100	192	0	9	1	10	202	221	19	NA	22
	Mean	4.2	4.5	8.7	0.0	0.4	0.0	0.5	9.2	10.0	0.9	42.1
	S.D.	2.08	1.63	2.57	0.00	0.80	0.21	0.86	2.22	1.68	1.78	5.98
	S.E.	0.44	0.35	0.55	0.00	0.17	0.05	0.18	0.47	0.36	0.38	1.27
100	Total	91	87	178	0	6	0	6	184	197	13	NA	20
	Mean	4.6	4.4	8.9	0.0	0.3	0.0	0.3	9.2	9.9	0.7	42.0
	S.D.	2.09	2.21	1.74	0.00	0.92	0.00	0.92	1.54	1.39	0.99	3.79
	S.E.	0.47	0.49	0.39	0.00	0.21	0.00	0.21	0.34	0.31	0.22	0.85
250	Total	88	117	205	1	2	8	11	216	236	20	NA	23
	Mean	3.8	5.1	8.9	0.0	0.1	0.3	0.5	9.4	10.3	0.9	41.4
	S.D.	1.67	1.70	1.83	0.21	0.29	0.71	0.85	2.13	2.12	0.92	4.57
	S.E.	0.35	0.36	0.38	0.04	0.06	0.15	0.18	0.44	0.44	0.19	0.95
1000	Total	88	97	185	0	3	3	6	191	205	14	NA	20
	Mean	4.4	4.9	9.3	0.0	0.2	0.2	0.3	9.6	10.3	0.7	37.7*
	S.D.	1.10	1.46	1.33	0.00	0.37	0.37	0.57	1.32	1.55	0.98	6.35
	S.E.	0.24	0.33	0.30	0.00	0.08	0.08	0.13	0.29	0.35	0.22	1.42

* = Significantly different from the control group at 0.05

NA = Not applicable

Mean Number of Viable Fetuses; Mean Number of Implantation Sites; Mean Number of Corpora Lutea; Fetal Weights compared using Dunnett’s Test.

Table 8. Summary of Litter Proportions of Malformations (% per Litter)
Dose Group (mg/Kd/day)		0 (Control)	100	250	500
Number of Litters Examined		22	20	23	20
Total Malformations
Percent per Litter with External Malformations	Mean	1.0	0.0	0.0	1.4
	S.D.	3.27	0.00	0.00	3.33
	S.E.	0.70	0.00	0.00	0.74
Percent per Litter with Soft Tissue Malformations	Mean	2.9	0.5	0.8	0.0
	S.D.	8.25	2.03	2.75	0.00
	S.E.	1.76	0.45	0.57	0.00
Percent per Litter with Skeletal Malformations	Mean	1.2	1.6	0.4	3.0
	S.D.	3.77	5.04	2.09	7.03
	S.E.	0.80	1.13	0.43	1.57
Total Percent per Litter with Malformations	Mean	5.0	2.1	1.3	4.0
	S.D.	10.62	6.73	4.50	7.17
	S.E.	2.26	1.51	0.94	1.60

Modified Statistics Used

Table 9. Summary of Fetuses and Litters with Variations (Absolute No.)
Dose Group (mg/Kg/day)	Fetuses				Litters
	0 (Control)	100	250	500	0 (Control)	100	250	500
Number Examined Externally	192	178	205	185	22	20	23	20
Excessive Fat Pads	1	0	0	0	1	0	0	0
Number Examined Viscerally	192	178	205	185	22	20	23	20
Major Blood Pressure Variation	20	15	14	19	8	7	9	9
Retrocaval Ureter	7	2	2	1	4	1	2	1
Accessory Spleen (S)	29	21	20	23	15	8	11	11
Heart Extra Papillary Muscle	4	3	4	3	2	2	3	2
Spleen – Small	0	0	0	3	0	0	0	2
Gall Bladder – Absent or Small	3	6	1	2	3	3	1	1
Liver – Accessory Lobule (s)	1	0	0	0	1	0	0	0
Spleen – Pale	2	0	0	6	1	0	0	3
Renal Papillae(s) Not Developed and/or Distended Ureters	1	0	0	0	1	0	0	0
Liver – Pale	0	0	0	1	0	0	0	1
Number Examined Skeletally	192	178	205	185	22	20	23	20
13^thRudimentary Rib(s)	36	28	41	22	15	13	18	15
13^thFull Rib(s)	64	64	52	48	18	16	18	14
27 Presacral Vertebrae	10	20	5	7	7	9	3	5
Sternebra(E) Malaligned (slight or moderate)	2	7	4	3	2	6	4	3
Sternebra(E) #5 and/or #6 Unossified	31	27	34	28	14	13	13	10
Hyoid Arch (es) Bent	4	3	2	3	4	2	2	3
Extra Site of Ossification Anterior to Sternebrae(E) # 1	7	0	8	2	4	0	7	2
Sternebrae with Thread like Attachment	1	3	0	2	1	2	0	1
7^thCervical Ribs	1	1	5	1	1	1	4	1
Pubis Unossified	0	0	0	3	0	0	0	1
Vertebral Centra not Fully Ossified	0	1	0	0	0	1	0	0
Accessory Skull Bone(s)	0	0	1	2	0	0	1	2
7^thSternebrae	0	0	0	1	0	0	0	1

Table 10. Summary of Fetuses and Litters with Malformations (Absolute No.)
Dose Group (mg/Kg/day)	Fetuses				Litters
	0 (Control)	100	250	500	0 (Control)	100	250	500
Number Examined Externally	192	178	205	185	22	20	23	20
Short-tail	0	0	0	1	0	0	0	1
Carpal and/or Tarsal Flexure	1	0	0	0	1	0	0	0
Polydactyly	1	0	0	0	1	0	0	0
Exencephaly with or without Open Eyelid	0	0	0	1	0	0	0	1
Macroglossia	1	0	0	0	1	0	0	0
Malpositioned Umbilicus	0	0	0	1	0	0	0	1
Number Examined Viscerally	192	178	205	185	22	20	23	20
Lungs – Lobular Agenesis	4	0	2	0	2	0	2	0
Vestigial Pulmonary Trunk	1	1	0	0	1	1	0	0
Bulbous Aorta	2	1	0	0	2	1	0	0
Interventricular Septal Defect	0	1	0	0	0	1	0	0
Heart – Three Chambered	1	0	0	0	1	0	0	0
Heart – Small Ventricle	1	0	0	0	1	0	0	0
Number Examined Skeletally	192	178	205	185	22	20	23	20
Vertebral Anomaly with or without associated Rib Anomaly	1	1	0	2	1	1	0	2
Rib Anomaly	0	0	1	0	0	0	1	0
Coastal Cartilage Anomaly	1	1	1	1	1	1	1	1
Sternebra (E) Malaligned (Severe)	0	1	0	3	0	1	0	3
Sternoschisis	0	0	0	1	0	0	0	1
Vertebral Centra Anomaly	0	0	0	1	0	0	0	1
Total number with malformations
External	2	0	0	3	2	0	0	3
Soft Tissue	5	1	2	0	3	1	2	0
Skeletal	2	3	1	6	2	2	1	4
Combined	9	4	3	8	6	2	2	6

Conclusions:

The no-observed-adverse-effect level (NOAEL) for maternal toxicity and embryo/fetal developmental toxicity was 250 mg/kg bw/day.

Executive summary:

Only screening reproductive/developmental toxicity 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 Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. The test substance, dipropyleneglycol dibenzoate (DPGDB), in the vehicle (0.5% carboxymethylcellulose in deionized water) was administered orally by gavage to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from Gestation Days 7–28. Dosage levels were 100, 250, and 500 mg/kg/day administered at a dose volume of 5 mL/kg. Adverse effects on maternal survival, mean body weight changes, and food consumption were noted in the 500 mg/kg/day group; therefore, a dosage level of 250 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on lower mean fetal weights at 500 mg/kg/day, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Since no severe developmental toxicity effects were observed and fetal weights reduction was considered to be due to maternal toxicity effect at 500 mg/kg/d group, the substance is not categorised as developmental toxicant under GHS classification criteria.

Test substance-related effects on survival were noted in the 500 mg/kg/day group, as 1 female was euthanized in extremis on Gestation Day 26 following a severe body weight loss and markedly reduced food consumption, with corresponding incidences of decreased defecation noted at the daily examinations. Adverse clinical observations of rales, labored respiration, decreased respiration rate, and a thin body were also noted for this female at the daily examinations and/or postdosing observations. An additional female in the 500 mg/kg/day group aborted on Gestation Day 25 and was subsequently euthanized. At necropsy, neither female had remarkable macroscopic findings. There were no other test-substance related effects on survival. In the 100 and 500 mg/kg/day groups, 3 and 1 females, respectively, were found dead or euthanized in extremis during Gestation Days 13–26; following macroscopic/microscopic examination, the cause of mortality/moribundity of these females were attributed to or considered likely related to intubation error. All other females survived to the scheduled necropsy. In the 500 mg/kg/day group, there was a slight increase in the incidence of decreased defecation at the daily examinations, which was considered secondary to the reduced mean food consumption noted in this group. Test substance-related lower mean body weight gains or body weight losses were noted in the 500 mg/kg/day group generally during Gestation Days 13–29, resulting in a lower mean body weight gain when the entire treatment period (Gestation Days 7–29) was evaluated compared to the control group. Mean food consumption was also lower in this group during Gestation Days 13–29, which resulted in slightly lower mean food consumption for the overall dosing period (Gestation Days 7–29), and corresponded to the decreased mean body weight gains noted in this group. In addition, mean net body weight loss was also noted compared to the control group at 500 mg/kg/day. Although mean body weights in the 500 mg/kg/day group were comparable to the control group throughout the treatment period, these changes were still considered adverse as they led to the mortality or abortion of 2 females within the group. Mean net body weight and gravid uterine weight were comparable to the control group at 500 mg/kg/day. In the 100 and 250 mg/kg/day groups, mean body weights, body weight changes, net body weights, net body weight changes, gravid uterine weights, and food consumption were unaffected by test substance administration. Hematology and serum chemistry parameters were unaffected by test substance administration at all dosage levels.

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance. Test substance-related adrenal cortical hypertrophy was noted for females in the 100, 250, and 500 mg/kg/day groups; however, due to the minimal to mild severity and the lack of other related microscopic and gross findings, this finding was considered non adverse. There were no other test-substance-related microscopic observations at any dosage level. Mean fetal body weights (male, female, and combined) were 8.9% to 11.8% lower in the 500 mg/kg/day group compared to the control group, and were considered test substance-related and adverse. Mean fetal body weights in the 100 and 250 mg/kg/day groups were similar to the control group. Intrauterine survival was unaffected by test substance administration at all dosage levels. No test substance-related effects on fetal morphology were noted at any dosage level.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 1998 - 31 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

See below ("Principles of method if other than guideline").

Deviations:

yes

Remarks:

Evaluation was made with knowledge of treatment group, as procedures are already in place to minimise bias during these portions of the study.

Qualifier:

according to guideline

Guideline:

other: Ministry of Agriculture, Forestry and Fisheries for Japan, Noh San No. 4200.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Economic Communities, Comission Directive 94/79EEC

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

The substance is mixture of benzoates and it is a clear liquid. The substance can be stored at ambient temparture.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England.
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 214 to 273g
- Fasting period before study: Not reported
- Housing: Cages consisting of stainless steel (acclimatisation and mating) or high-density polypropylene (gestation) bodies with lids and floors of stainless steel grid, suspended in batteries over trays covered with absorbent paper
- Diet: Free access to a commercially available pelleted laboratory animal diet
- Water: Tap water from the public supply was freely available.
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Nominally 21°C (Range 19 - 23°C)
- Humidity (%): Nominally 55% (range 40 - 70%)
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light.


IN-LIFE DATES: From: 2 December 1998 To: 31 December 1998

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the highest required concentration (200 mg/mL) the required amount of DPGDB was weighed out and mixed with a small amount of the vehicle. This mixture was quantitatively transferred to the mixing vessel with further portions of vehicle and adjusted to volume. Homogenisation of the final product was achieved using a mechanical blender such as a Silverson stirrer/emulsifier. Lower concentrations were prepared by serial dilution. The use of equipment containing polar rubber compounds or polyvinyl chloride was avoided during the preparation/dispensing of formulations as much as possible.

VEHICLE
- Justification for use and choice of vehicle (if other than water): DPGDB has poor water solubility
- Concentration in vehicle: 0, 50, 100, and 200 mg/mL (for dose levels 0, 250, 500, and 1000 mg/kg/day, respectively).
- Amount of vehicle (if gavage): 5 mL/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from formulations prepared for use during the first and last weeks of the dosing period for determination of achieved concentrations of DPGDB. On each occasion of sampling four samples (nominally 1 mL accurately weighed) were taken from each formulation; 2 assays were performed from each test group and one assay for the control group. The remainder of the samples were frozen (nominally -20°C) as contingency for analysis if any result required confirmation. These samples were taken in the Pharmacy department using the types of glass syringes and rubber catheters used to dose the animals. This precaution was taken to provide assurance that contact between the dose formulations and the rubber catheter did not affect the achieved concentrations of DPGDB.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one-to-one
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of
gestation.

Duration of treatment / exposure:

13 Days (Days 6 to Day 19 after, inclusive)

Frequency of treatment:

Once per day administration

Duration of test:

20 Days

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Eighty eight females showing unequivocal evidence of mating were allocated to group and cage position in sequence thus ensuring that animals mated on any one day were evenly distributed amongst the groups

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Pre-dosing, on return of the animal to home cage, after dosing each group, 1 to 2 hours after completion of dosing, and as late as possible during the working day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily (as above)


BODY WEIGHT: Yes
- Time schedule for examinations: Weighed on days 0, 3, 6 to 17 inclusive and 20 after mating


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No - calculated as g/rat/day



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: The organs of the reproductive tract, complete with ovaries.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (Uterus with cervix)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: The number and distribution of fetuses in each uterine horn

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Statistical tests employing analysis of variance followed by an inter group comparison with the Control were performed on the following parameters:
Bodyweight change, bodyweight change adjusted for gravid uterine weight, food consumption, litter data, litter weight, fetal weight and placental
weight.
Dependant on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams 1971/2) or non parametric tests (Kruskal-Wallis, Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate.
For litter data (excluding fetal, litter and placental weights) and implantation loss, due to the preponderance of non-normal distributions, non-parametric tests are generally the most consistent and were routinely used.
All significant (i.e. p<0.05) inter-group differences from the Control are reported only where supported by a significant analysis ofvariance (i.e. p<0.05).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

one animal showed salivation after the second dose (Day 7) at 1000 mg/kg/d but by the fifth dose (Day 10) the majority of animals were affected. The pattern was similar at 500 mg/kg/d except that the day of first occurence was Day 10 (fifth dose). At 250 mg/kg/d, the occurrence of salivation was much more transitory, with no animals being affected on more than four cosecutive day.

Mortality:

no mortality observed

Description (incidence):

There were no deaths observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment on absolute or adjusted bodyweight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on treatment on food intake

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

No treatment related effects were observed.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no effects of treatment were indicated by the extent of pre and post implantaion loss. The mean percentage value for pre implantaion loss for control group was 7.1, 6.6 for Group 2, 5.2 for Group 3 and 5.4 for Group 4. No statistical significance values were acheived. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no effects of treatment were indicated by resorption. The mean percentages for total resorption was 0.9, 0.8, 0.8 and 1.0 for Group 1,2,3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no effects of treatment were indicated by early or late resorption. The mean percentages for early resorption was 0.9, 0.7, 0.8 and 1.0 for Group 1,2,3 and 4. The mean percentages for early resorption was 0.0, 0.0, 0.0 and 0.0 for Group 1,2,3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Dead fetuses:

no effects observed

Description (incidence and severity):

No effects of treatment on prenatal survival and the number of live fetuses were observed. The mean group values for live fetus were similar in all groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

22 pregnant females per group were evaluted after treatment of females from day 6 to day 19.

Description (incidence and severity):

1 (0 mg/kg/d) 2(250 mg/kg/d)3(500 mg/kg/d)4 (1000 mg/kg/d)

pregnant females 22 22 22 22
evalated pregnant females 22 22 22 22


The group mean values for litters were

1 2 3 4
Corpora lutea 16.8 16.3 16.5 16.0
Implantations 15.4 15.4 15.6 15.1
Resorptions 0.9 0.8 0.8 1.0
Live fetus 14.5 14.6 14.9 14.1
Sex ratio (%) male 49.7 51.5 49.6 49.1
female 50.3 48.5 50.4 50.9
weight of fetus (g) male 3.88 3.94 3.87 3.84
female 3.71 3.75 3.68 3.66

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical signs associated with treatment were restricted to transient post-dosing salivation.
There was no effect of treatment on absolute or adjusted bodyweight gain.
There was no effect of treatment on food intake.
There were no findings considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Basis for effect level:

clinical signs

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The overall fetal body weights were comparable to control and were not statistically significant in any group in comparison to control. The overall mean (g) weight was 3.79 in control 1 and 3.85, 3.77 and 3.74 gm in Group 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was 49.7, 51.5, 49.6 and 49.1% in Group 1, 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Description (incidence and severity):

There were no effects of treatment were indicated in litter weights. The mean (g) litter weight was 54. 94 in control 1 and 56.14, 56.01 and 52.64 gm in Group 2, 3 and 4. The Group 1 is control (0 mg/kg/d) and group 2 is 250 mg/kg/d and group 3 is 500 mg/kg/d and group 4 is 1000 mg/kg/d dose group.

Description (incidence and severity):

The live young male and female percentage in Group 1,2,3 and 4 were 7.2, 7.4, 7.4 and 7 and 7.3, 7.2,7.5 and 7.1. The treated groups were comparable to the control group.

External malformations:

no effects observed

Description (incidence and severity):

There were no effect of treatment on the incidence of external malformations were observed.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

at 1000 mg/kg/d, the skeletal examination revealed a small but definite increase in the incidence of cervical ribs compared with the controls and the background historical control data. Therefore the findings of this skeletal anomaly is considered to be related to treatment. At 500 and 250 mg/kg/d, the incidence of fetuses with cervical ribs was within the recent background control data and there was no clear dosage relationship, their occurence at these dosages is likely to be coincidental and unrelated to the treatment.

An association between treatment at 1000 and 500 mg/kg bw/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.
The increase in cervical ribs at 1000 mg/kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kg bw/day and there was no concomitant change in vertebral configuration.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

There were treatment related minor visceral anomalies were obsreved.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An association between treatment at 1000 and 500 mg/kg/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

The increase in cervical ribs at 1000 mg/kg/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kgl/day and there was no concomitant change in vertebral configuration.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

skeletal malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The no-adverse-effect-level for maternal toxicity was concluded to be 1000 mg/Kg bw/day
The no-observed adverse effect level for all aspects of pre natal development was 500 mg/Kg bw/day.

Executive summary:

Only screening reproductive/developmental toxicity 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 pre-natal development study in rats was conducted to determine the effect of the test material DPGDB when administered during and beyond the organogenesis phase of gestation. The study was conducted according to Japanese, US EPA and OECD test guidelines, and in compliance with GLP.

Groups of 22 female rats were selected after mating, and were dosed by oral gavage with corn oil fortified with the test material between day 6 and day 19 of gestation. Dose levels examined were 0 (vehicle control), 250, 500, and 1000 mg/kg bw/day.

An association between treatment at 1000 and 500 mg/kg bw/day and the greater number of fetuses with incomplete ossification of the 5th and or 6th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

The increase in cervical ribs at 1000 mg/kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/kg bw/day and there was no concomitant change in vertebral configuration.

Salivation after dosing was observed at all dosages of benzoflex, the incidence was dose related but this finding was not considered to be of toxicological importance. At 1000 mg/kg/d, there were no detectable signs of maternal toxicity, there were no maternal deaths and all females had a live litter scarifice. It was concluded that the 1000 mg/kg/d is the NOAEL for maternal toxicity.There were no treatment related effects observed at prenatal survival or growth. At 1000 mg/kg/d, treatment related small but definite increase in the number of fetus with cervical ribs were observed. The no-observed adverse effect level for all aspects of pre-natal development is concluded to be 500 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimish 1 rating, study conducted in a GLP compliant lab according to current test Guidelines

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Only screening reproductive/developmental toxicity 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.

 

In a key read across OECD Guideline 414 pre-natal development study in rats (Huntingdon, 2000c; Klimisch score = 1), conducted to determine the effect of the test material (DPGDB) when administered during and beyond the organogenesis phase of gestation, groups of 22 female rats were selected after mating, and were dosed by oral gavage with corn oil fortified with the test material between day 6 and day 19 of gestation. Dose levels examined were 0 (vehicle control), 250, 500, and 1000 mg/Kg bw/day. According to preliminary results obtained in rats in a dose range-finding study (HLS 2000, VCL313/980305), doses up to 1500 mg/Kg bw/day during gestation days 6 to 19 gave no adverse effect on dams or foetuses, but maternal toxicity was observed at the highest dose. The highest dose used in the main study was therefore 1000 mg/Kg bw/day.

 

In the rat study, an association between treatment at 1000 and 500 mg/Kg bw/day and the greater number of fetuses with incomplete ossification of the 5^th and/or 6^th sternebrae cannot be discounted particularly since a delay in ossification would be expected to be the most sensitive marker of an effect on pre-natal development where treatment has continued through to the day before sacrifice (treatment period: Days 6 to 19 of gestation). The assessment of fetal ossification on Day 20 of gestation represents a snapshot in time as the ossification will continue as the animals grow and mature. Although the relationship of these findings to treatment is uncertain they are considered to be transient in nature rather than representing permanent structural changes and therefore are considered to be of no long-term toxicological importance.

 

The increase in cervical ribs at 1000 mg/Kg bw/day is considered to be of greater toxicological significance as it occurred at a dosage which has not produced any detectable signs of maternal toxicity however cervical ribs were only found in a small number of fetuses (10/155) at the limit dosage of 1000 mg/Kg bw/day and there was no concomitant change in vertebral configuration.

 

Salivation after dosing was observed at all dosages of the test material; the incidence was dose related but this finding was not considered to be of toxicological importance. At 1000 mg/Kg bw/day, there were no detectable signs of maternal toxicity, there were no maternal deaths and all females had a live litter sacrifice. It was concluded that the 1000 mg/Kg bw/day is the NOAEL for maternal toxicity. There were no treatment related effects observed at prenatal survival or growth. At 1000 mg/Kg bw/day, treatment related small but definite increase in the number of fetuses with cervical ribs were observed. The no-observed adverse effect level for all aspects of pre-natal development was concluded to be 500 mg/Kg bw/day.

 

In a key read across OECD Guideline 414 pre-natal development study in rabbits (Charles River Laboratories, 2018a; Klimisch score = 1), the test material (dipropyleneglycol dibenzoate (DPGDB)), in the vehicle (0.5% carboxymethylcellulose in deionized water) was administered orally by gavage to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits once daily from Gestation Days 7–28. Dosage levels were 100, 250, and 500 mg/Kg bw/day administered at a dose volume of 5 mL/Kg.

 

No fetal malformations were attributed to the test substance. Other fetal developmental variations occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges, and therefore were not attributed to the test substance. Adverse effects on maternal survival, mean body weight changes, and food consumption were noted in the 500 mg/Kg bw/day group; therefore, a dosage level of 250 mg/Kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity.

 

Based on lower mean fetal weights at 500 mg/Kg bw/day, a dosage level of 250 mg/Kg bw/day was considered to be the NOAEL for embryo/fetal developmental toxicity when dipropyleneglycol dibenzoate was administered orally by gavage to time-mated New Zealand White rabbits. Importantly, a 10.5% decrease in fetal body weight in the 500 mg/Kg bw/day dosage group reflects the 17% decrease in feed consumption in the dams during the fetal period and therefore this reduced fetal body weight is related to the maternal toxicity that was observed at that dose level.

In a key OECD Guideline 422 combined repeat dose / reproductive and developmental toxicity screening study (Huntingdon, 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.

There was a slight decrease in live birth index and a small increase in the number of offspring dying between birth and Day 7 of age for the group receiving 1000 mg/Kg bw/day. Male and female offspring body weight on Day 1 of age was low at 1000 mg/Kg bw/day; this wasdespite a tendency towards a longer gestation length which was probably secondary to restricted intra uterine growth. Offspring body weight gain thereafter remained slightly low at 300 or 1000 mg/Kg bw/day. Macroscopic examination at scheduled termination on Day 7 of age confirmed this with, a number of offspring in the 1000 mg/Kg bw/day group noted to be of thin build and with no milk in stomach. As the effects on offspring survival were limited to the 1000 mg/Kg bw/day group, the effects at 300 mg/Kg bw/day were considered not to be adverse at the degree observed.

 

Based on the results observed, the NOAEL for developmental toxicity was determined to be 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality.

Toxicity to reproduction: other studies

Description of key information

In a vaginal cornification/uterine weight bioassay, DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose (Bioqual Inc 1997, Vel 001-97).

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Remarks:

Evaluation for Estrogenic Activity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study conducted to GLP and although no International Test guideline available

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:

no guideline available

Principles of method if other than guideline:

Adult ovariectomised rats (8 per group) were treated orally for 7 days (4 test groups + 1 vehicle control + 3 groups for positive control Diethylstilbestrol)
All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification.
All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.

GLP compliance:

yes

Type of method:

in vivo

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY, USA.
- Age at study initiation: (P) x 9 wks
- Weight at study initiation: (P) Females: 170 -200 g
- Fasting period before study: Not applicable
- Housing: Individually in stainless steel hanging wire cages.
- Diet: Purina rodent chow (#5001), ad libitum
- Water: Mains (Montgomery County) tap water, ad libitum
- Acclimation period: 3 days prior to ovariectomy. Following a 6-7 days post surgery recovery period, evaluation of cell types was conducted daily
for one week prior to initiation of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Ranged 61°F to 76°F
- Humidity (%): Ranged 41% - 69%
- Photoperiod (hrs dark / hrs light): 10 hours dark/14 hours light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No details are available on the method of preparation of the dose solutions


VEHICLE
- Justification for use and choice of vehicle (if other than water): Test substance is poorly soluble in water
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): Mazola corn oil purchased from a local supermarket.
- Purity: Not stated but food grade

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Dosed once per day

Frequency of treatment:

Daily

Duration of test:

7 days

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1500 mg/kg/day
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

8 females per group

Control animals:

yes, concurrent vehicle

other: Positive control: ethylstilbestrol (DES) at 2.5, 5 & 10 µg/kg/day

Details on study design:

Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control diethylstilbestrol) were orally dosed for 7 days. All rats were observed daily for clinical signs and subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.

Statistics:

The occurrence of vaginal cornification was expressed as the number of rats showing vaginal cornification (for one or more days) over the number of rats treated. A z-test of proportions was used to compare the test groups to the control animals. The group means ± SE were calculated for body weights on day 0 and day 7 and a parametric one way analysis of variance (ANOVA) was performed. For a significant F value (p< 0.05), a Student-Newman-Keuls multiple range test was performed to determine differences among groups. Group means ± SE were also calculated for the uterine weights and uterine weight/final body weight ratios These data were compared using a non parametric one way ANOVA on ranks followed by Dunn's comparison to the designated control group.

Key result

Dose descriptor:

other: estrogenic activity

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on induction of vaginal cornification compared to vehicle & positive controls

Key result

Dose descriptor:

other: uterine weight increase

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on increase in uterine weight to final bodyweight

Clinical signs of toxicity were apparent in 8 out of 8 ovariectomized adult rats treated with DPGDB at 2000 mg/kg/day.

These observations included rapid and or shallow breathing, tremors, piloerection, thinness, lethargy, discharge around the eyes, nose and mouth - often red in appearance, hypothermia and ataxia. One of the eight rats was found dead and two moribund animals were euthanized. The necropsy findings for these three rats included weight loss and abnormalities in the gastrointestinal tract. At necropsy no gross abnormalities were observed in the 5 surviving rats.

One rat dosed at 1500 mg/kg/day had a scruffy coat but survived the 7 days of dosing and no gross abnormalities were noted at necropsy. All other animals appeared normal during the study and at necropsy.

With respect to estrogenic activity, DES (the positive control) stimulated a dose dependent induction of vaginal cornification. The 2.5 µg DES/kg/day dose was a threshold dose for this endpoint, in that the onset for the cornification response was of longer duration and more variable than in the 5.0 and 10.0 µg/kg/day groups. In addition, unlike the mid and high dose DES groups, not all rats in the low dose DES group gave a vaginal cornification response. Further, in the low dose DES group the number of rats cornified per number treated was significantly increased (p<0.05) only on days 4, 6 and 7 of the study, whereas for the mid and high dose DES groups this endpoint was significantly increased (p<0.05) from study days 3 through 7 and days 2 through 7, respectively.

In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 or 2000 mg/kg/day x 7 days. Hence by this endpoint DPGDB did not sjhow estrogenic activity.

DES tended to suppress body weight gain in a dose-dependent manner over the 7 day dosing period. Suppression of body weight gain or loss is a well known biological effect of potent estrogens. Only the high dose of DPGDB tended to decrease bodyweight gain but this effect was not significantly different for animals surviving 7 days of dosing.

DES induced a dose-dependent increase in uterine weight and uterine weight to final body weight ratio. Both parameters were increased significantly (p<0.05) at all dose levels of DES as compared to the vehicle control group. In contrast DPGDB did not stimulate an uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/ kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.

Conclusions:

Dipropylene glycol dibenzoate did not possess estrogenic activity up to and including the maximally tolerated dose.

Executive summary:

Only screening reproductive/developmental toxicity 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 study was performed to investigate whether the test substance, dipropylene glycol dibenzoate, had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay. The study was conducted to GLP but no formal international test guidelines were applicable.

Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control Diethylstilbestrol (DES)) were orally dosed for 7 days. All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.

DES, the positive control, resulted in a dose dependent induction of vaginal cornification. In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/kg/day x 7 days. Hence by this endpoint the test compound showed no estrogenic activity.

DES also induced a dose dependent increase in uterine weight and uterine weight to final body weight ratio. Conversely, DPGDB did not stimulate a uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/ kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.

Additional information

A study was performed to investigate whether the test substance, dipropylene glycol dibenzoate (DPGDB), had the potential for estrogenic activity when evaluated in a seven day rat vaginal cornification/uterine weight bioassay (Bioqual Inc., 1997; Klimisch Score = 1). The study was conducted to GLP but no formal international test guidelines were applicable.

 

Eight ovariectomised rats per group (4 test groups + 1 vehicle control + 3 groups positive control Diethylstilbestrol (DES)) were orally dosed for 7 days. All rats were subjected to vaginal lavage daily during the 7 day dosing period in order to detect vaginal cornification. All surviving rats were euthanized 24 hours after the final dose when final bodyweights and uterine weights were recorded.DES, the positive control, resulted in a dose dependent induction of vaginal cornification. In contrast DPGDB did not induce vaginal cornification at doses of 500, 1000, 1500 and 2000 mg/Kg/day x 7 days. Hence by this endpoint the test compound showed no estrogenic activity.

 

DES also induced a dose dependent increase in uterine weight and uterine weight to final body weight ratio. Conversely, DPGDB did not stimulate a uterine weight increase or an increase in the uterine weight to final body weight ratio at doses of 500, 1000, 1500 and 2000 mg/Kg/day x 7 days.

Collectively, these data demonstrate that DPGDB did not possess estrogenic activity up to and including the maximally tolerated dose.

Justification for classification or non-classification

Only screening reproductive/developmental toxicity 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.

 

PGDB

In the reproductive/developmental toxicity screening test (Huntingdon Life Sciences, 2014a), the NOAEL for systemic toxicity of the parents and reproductive and developmental toxicity was 300 mg/Kg bw/day, based on the low offspring growth and small increase in post-natal offspring mortality. The effects in pups were minimal and they started recovering by Day 7. These effects happened at the highest dose at which the mother showed some effects of systemic toxicity (low haematocrit, low calcium and albumin levels and high motor activity).

 

DPGDB

Based on the usual criteria for classification for developmental toxicity such as treatment related increases in embryofoetal mortality with increased resorptions, increase in congenital malformations, decreased fetal bodyweight etc., it is clear that no classification is justified. The only effects observed that require further consideration are the small increase in anomalies, viz. cervical ribs, and a small increase in retarded ossification of the sternebrae. 

 

With respect to retarded ossification, this effect is proceeding at a high rate during the last few days of gestation and a wide range of degree of ossification of fetuses is expected when the fetuses are delivered one or two days before expected parturition. In the control fetuses in the rat study (Huntingdon, 2000a), incomplete ossification of 5^th and/or 6^th sternebrae was present in 77/158 (49%) of fetuses. The mid and high dose groups showed the same effect in 104/165 (64%) and 104/155 (67%) of foetuses, respectively. This was a very small increase in a very common variation which would almost certainly disappear by term and was considered to be of no lasting toxicological significance.

 

With respect to the presence of cervical ribs, this effect was observed in 10/155 (6.5%) fetuses from 6/22 litters in the top dose group. In the mid and low dose groups, 1 fetus from 2 litters was affected in each dose group. The incidence in the top dose group was greater than in the concurrent controls and greater than has been seen in historical control groups. In historical control groups, data are available for 13 studies between 1997-1999, and in 7 of the 13 studies control litters contained fetuses with cervical ribs. The number of affected fetuses per litter ranged from 0-4 per litter. Because of the higher number of affected fetuses in the top dose group in the developmental toxicology study, that also exceed the historical control data from this laboratory, and also above the historical control data for the same strain of rat published by Charles River who showed that in Crl CD SD rats, the range for cervical ribs in 2800 control litters was zero to 16.7% of litters (0- 3.7% of fetuses) (Barbeau et al. 2008), this finding was considered to be related to treatment.

 

However, cervical ribs are not regarded as a malformation, but as an anomaly that occurs relatively frequently in rats of this strain. The most common type of this anomaly is reversible, disappearing postnatally, though in some cases can persist postnatally (Chernoff and Rogers, 2004). It is a common response to stress in the dams. The presence of a low incidence as in the rat study at toxic dose levels of 1000 mg/Kg bw/day is not normally regarded as any great toxicological significance. There was no evidence reported of gross toxicity in the dams in this study, but examination of the data available from the range-finding study showed that the dose of 1500 mg/Kg bw/day for 13 days was near to the LD₅₀ for the substance with half the animals becoming moribund within the final two days of exposure. This is further supported by a study quoted in the report of an earlier study in the same strain of female rats dosed with 2000 mg/Kg for up to 7 days which resulted in the death of 3 of 8 rats. The onset of toxic signs in these studies did not appear until after several days of intense salivation followed by sudden onset of bodyweight loss and then the animals became moribund within a further two days or so. At PM changes in the consistency of the gut contents was noted as the only finding. Thus, although there were no deaths at 1000 mg/Kg bw/day after 13 days of dosing in the rat study, there were clear signs of marked salivation by that time and no detailed PM examination of gut contents was reported. It is not unreasonable to assume that there was in fact maternal stress at this high dose level.

 

In deciding whether the presence of a small number of minor anomalies at a high dose level of 1000 mg/Kg bw/day would qualify for a substance to be classified under the CLP Regulation it is useful to look at the Guidance published by ECHA (ECHA, 2009) in relation to minor effects observed in studies. In paragraph 3.7.2.4.1., it is stated that “Development of the offspring throughout gestation and during the early postnatal stages can be influenced by toxic effects in the mother either through non-specific mechanisms related to stress and the disruption of maternal homeostasis, or by specific maternally-mediated mechanisms. In the interpretation of the developmental outcome to decide classification for developmental effects it is important to consider the possible influence of maternal toxicity. This is a complex issue because of uncertainties surrounding the relationship between maternal toxicity and developmental outcome. Expert judgement and a weight of evidence approach, using all available studies, shall be used to determine the degree of influence that shall be attributed to maternal toxicity when interpreting the criteria for classification for developmental effects.” In paragraph 3.7.2.4.3., it is further stated that “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity.” 

 

There is considerable discussion in the Guidance in relation to maternal toxicity and it states that even in the presence of considerable maternal toxicity classification (perhaps in Category 2) should be considered where there is “a significant toxic effect in the offspring, e.g. irreversible effects such as structural malformations, embryo/foetal lethality, significant post-natal functional deficiencies.”  Since no treatment related malformations or embryo-fetal lethality or any other severe irreversible effects were observed in the developmental toxicology study even at the highest dose level of 1000 mg/Kg bw/day, but only a small increase in minor anomalies, it is concluded that a classification for developmental toxicity for DPGDB under the CLP Regulations is not warranted.

 

Additionally, a Prenatal Developmental GLP Toxicity Study was recently conducted according to test guideline 414. Based on lower mean fetal weights at 500 mg/Kg bw/day, a dosage level of 250 mg/Kg bw/day was considered to be the NOAEL for embryo/fetal developmental toxicity when DPGDB was administered orally by gavage to time-mated New Zealand White rabbits. No fetal malformations were attributed to the test substance. Other fetal developmental variations occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges, and therefore were not attributed to the test substance. Importantly, decrease in fetal body weight in the 500 mg/Kg bw/day dosage group reflects the decrease in feed consumption in the dams during the fetal period and therefore this reduced fetal body weight is related to the maternal toxicity that was observed at that dose level. There were no severe developmental effects or fetal toxicity was observed at the any dose levels, therefore DPGDB was not classified under GHS classification.

 

Based on available substance data and read across data from Dipropylene glycol dibenzoate (DPGDB), a structural analogue which is also not classified for reproductive toxicity, it is concluded that PGDB does not warrant classification as a reproductive or developmental toxicant under the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.