Oxydipropyl dibenzoate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

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:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

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

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

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3300 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
10000 ppm
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:

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.

Effect on fertility: via oral route

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

chronic

Species:

rat

Additional information

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 (HLS 2001, VCL 316/004080). According to preliminary results obtained in rats in a dose range-finding study (HLS 2006, VCL315/990088), concentrations of DPGDB in diet at 25000 ppm during two weeks before mating, during gestation and lactation gave evidence of toxicity in dams (mortality, clinical signs). This preliminary study suggested that an upper dietary concentration of DPGDB between 7500 and 15000 ppm would be appropriate in a main two generation study of reproductive performance.

 

In the main study, 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. 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 10000ppm 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-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/d).

Short description of key information:
In a two generation study, conducted according to OECD and EPA test guidelines, and in compliance with GLP, realised in rats to assess the effects on reproductive performance of the test material DPGDB (HLS 2001, VCL 316/004080), the NOAEL for F0 and F1 parent animals and the NOAEL for survival and growth of the offspring were both considered to be 10000 ppm in diet.

Effects on developmental toxicity

Description of key information

 A Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. 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. Importantly, a 10.5% decrease in fetal body weight in the 500 mg/kg/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

Link to relevant study records

Reference

Reference 1

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

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/kgbw/day
The no-observed adverse effect level for all aspects of pre natal development was 500 mg/kgbw/day.

Executive summary:

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.

Reference 2

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

Dipropyleneglycol dibenzoate, Clear, colorless liquid. The purity of the test substance was > 99.0%. The test substance was stored at room temperature (18°C to 24°C) and was considered stable under this condition.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

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. The animals were approximately 7 months old upon receipt.All rabbits were housed during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 61°F to 71°F (16°C to 22°C) and 30% to 70%, respectively.The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

The day on which confirmation of mating is made was designated as Gestation Day 0. Time mated rabbits judged to be suitable test subjects and meeting acceptable body weight requirements were assigned to the study at random using a computer program. Gestation Day 0 body weights were used for randomization. The animal numbers and corresponding body weights will be entered into the WIL Toxicology Data Management System . A printout containing the animal numbers and individual group assignments will be generated based on body weight stratification into a block design. The rabbits will then be arranged into the groups according to the printout. The control group and 3 test substance groups will consist of 24 rabbits each.Time-mated rabbits will be received by Charles River on Gestation Days 1, 2, 3, and 4.

Route of administration:

oral: gavage

Vehicle:

other: carboxymethylcellulose

Remarks:

carboxymethylcellulose

Details on 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. Following administration of each dose, the catheter was flushed with 5 mL of deionized water to ensure delivery of the entire dose.

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.:

100 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

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:

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. A concurrent control group composed of 24 time-mated females received the vehicle on a comparable regimen. The females were approximately 7 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. Clinical pathology evaluations (hematology and serum chemistry) were performed on all females that were euthanized in extremis or exhibited signs of toxicity, and on 5 females/group at the scheduled necropsy. On Gestation Day 29, a laparohysterectomy was performed on each surviving female. 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. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Maternal examinations:

Clinical observations, body weights, and food consumption were recorded at appropriate intervals. Clinical pathology evaluations (hematology and serum chemistry) were performed on all females that were euthanized in extremis or exhibited signs of toxicity, and on 5 females/group at the scheduled necropsy. On Gestation Day 29, a laparohysterectomy was performed on each surviving female.

Ovaries and uterine content:

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

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.

Historical control data:

Charles River Ashland historical control data was used.

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. (Attached table)

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, non-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 (Refrence: attached tables).





DOSE GROUP : 1 2 3 4
-------------------------------------------------------------------------------------------------------------------------------

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
NONGRAVID 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
NONGRAVID 0 0.0 0 0.0 0 0.0 0 0.0
GRAVID 0 0.0 2 100.0 0 0.0 1 100.0

Total females Gravid 22 91.7 23 95.8 23 95.8 23 95.8

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.

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 resoptions 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 (Refer attached tables) .

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).

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

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 (attached table in word for ref).

MALE FETAL WEIGHTS (gm) MEAN 42.8 (0 mg/kg/d), 42.4 (100 mg/kg/d), 41.8, (250 mg/kg/d), 39.0 (500 mg/kg/d)
FEMALE FETAL WEIGHTS (gm) MEAN 41.6 (0 mg/kg/d), 41.1 ( 100 mg/kg/d), 41.2 (250 mg/kg/d), 36.7** (500 mg/kg/d)
Combined weights (gm) 42.1 (0 mg/kg/d), 42.0 ( 100 mg/kg/d), 41.4 (250 mg/kg/d), 37.7** (500 mg/kg/d)

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 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 and Group 2 is 100 mg/kg/d group 3- 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 eefcts and values were comparable to controls.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The sex ratio was similar across all groups
M F
TOTAL 92 100 (0 mg/kg/d)
TOTAL 91 87 ( 100 mg/kg/d)
TOTAL 88 117 (250 mg/kg/d)
TOTAL 88 97 (500 mg/kg/d)

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.

SUMMARY OF FETUSES AND LITTERS WITH MALFORMATIONS

fetus litter
DOSE GROUP: 1 2 3 4 1 2 3 4
-------------------------------------------------------------------------------------------------------------------------------

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

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 250 MG/KG/DAY 4- 500 MG/KG/DAY

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.

SUMMARY OF FETUSES AND LITTERS WITH SKELETAL MALFORMATIONS
fetus litter
DOSE GROUP: 1 2 3 4 1 2 3 4
-------------------------------------------------------------------------------------------------------------------------------

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
COSTAL 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

1- 0 MG/KG/DAY 2- 100 MG/KG/DAY 3- 250 MG/KG/DAY 4- 500 MG/KG/DAY

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.


Dose group 1 2 3 4 1 2 3 4
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

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.

Description (incidence and severity):

TOTAL NUMBER WITH MALFORMATIONS
Dose group 1 2 3 4 (fetus) 1 2 3 4 (Litter)
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

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.

F E T U S E S L I T T E R S
DOSE GROUP: 1 2 3 4 1 2 3 4
-------------------------------------------------------------------------------------------------------------------

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 VESSEL 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
GALLBLADDER- 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 PAPILLA(E) NOT DEVELOPED AND/OR DISTENDED URETER(S) 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
13TH RUDIMENTARY RIB(S) 36 28 41 32 15 13 18 15
13TH FULL 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 STERNEBRA #1 7 0 8 2 4 0 7 2
STERNEBRAE WITH THREAD-LIKE ATTACHMENT 1 3 0 2 1 2 0 1
7TH CERVICAL RIB(S) 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


NUMBER EXAMINED SKELETALLY 192 178 205 185 22 20 23 20
ACCESSORY SKULL BONE(S) 0 0 1 2 0 0 1 2
7TH STERNEBRA 0 0 0 1 0 0 0 1

Details on embryotoxic / teratogenic effects:

SUMMARY OF FETAL DATA for IMPLANTATION SITES and CORPORA LUTEA
IMPLANTATION CORPORA
SITES LUTEA
Group 1 202 221
Group 2 184 197
Group 3 216 236
Group 4 191 205

Group 1- 0 MG/KG/DAY , Group 2- 100 MG/KG/DAY Group 3- 250 MG/KG/DAY Group 4- 500 MG/KG/DAY

The effects were not significantly different from the control group.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

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

Refrences: See attached tabels (Summary and indivisual data)

Conclusions:

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

Executive summary:

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.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

500 mg/kg bw/day

Additional information

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 (HLS 2000, VCL314/993005).

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 1500mg/kg/d 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 1000mg/kg/d.

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 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.

Toxicity to reproduction: other studies

Description of key information

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. 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/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. Importantly, a 10.5% decrease in fetal body weight in the 500 mg/kg/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.

Additional information

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.

Justification for classification or non-classification

Based on the usual criteria for classification for developmental toxicity such as treatment related increases in embryofetal 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 present study incomplete ossification of 5th and/or 6th sternebrae is present in 77/158 (49%) of fetuses. The mid and high dose groups show the same effect in 104/165 (64%) and 104/155 (67%) of fetuses respectively. This is a very small increase in a very common variation which would almost certainly disappear by term and is 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 were affected in each dose group. The incidence in the top dose group is 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 is 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 present study at toxic dose levels of 1000 mg/kg bw is not normally regarded as any great toxicological significance. There is no evidence reported of gross toxicity in the dams in this study, but examination of the data available from the range-finding study showedthe dose of 1500 mg/kg 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 after 13 days of dosing in the present study, there were clear signs of marked salivation by that time and no detailed PM examination of gut contents is 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 1000mg/kg bw 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, but only a small increase in minor anomalies, it is concluded that a classification for developmental toxicity for dipropylene glycol dibenzoate under the CLP Regulations is not warranted.

A recently conducted Prenatal Developmental GLP Toxicity Study was conducted according to test guideline 414. 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. 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/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 the substance was not classified under GHS classification.