Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 9-88 (Dipropylene glycol dibenzoate DPGDB)
- Physical state: A clear colourless liquid
- Analytical purity: Dipropylene glycol dibenzoate - 98.84 Area%
- Impurities (identity and concentrations):
Dipropylene glycol monobenzoate - 2.91 Area%
Propylene glycol dibenzoate 1 - 1.79 Area%
2- (2-Propylenoxy) 1-propyl benzoate - 0.29 Area%
- Lot/batch No.: 558260E1-4
- Storage condition of test material: Ambient temperature
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.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
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 FO males, terminal bodyweight was comparable in all groups. Absolute and bodyweight relative weights for the adrenal glands were marginally but significantly higher than in Controls at 10000 and 3300 ppm. There were no other significant differences in absolute or bodyweight relative organ weights.
Among FO females, terminal bodyweight was 94%, 97% and 93% of Controls in the 1000,3300 and 10000 ppm groups respectively. Absolute weight for the kidneys 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 adrenal glands and brain were significantly higher than in Controls; the difference in brain weight is considered to be of no toxicological importance since absolute brain weight was similar to Controls despite the slightly lower terminal bodyweight.
The higher absolute and/or bodyweight relative weights for the adrenal glands apparent among males and females at 10000 ppm and males at 3300 ppm are considered to be of doubtful biological significance since no treatment related findings were detected at microscopic examination.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic abnormalities detected at necropsy of the FO males or females that were considered to be as a result of treatment with Benzoflex" 9-88.
Neuropathological findings:
not examined
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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
10 000 ppm (nominal)
Sex:
male/female
Remarks on result:
other: Generation: F1 parents (migrated information)

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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.

Effect levels (F1)

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

Results: F2 generation

General toxicity (F2)

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.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.