Registration Dossier

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:
25 May 1999 to 19 February 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD test guidelines and in compliance with GLP

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
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 2-45 (Diethylene glycol dibenzoate DEGDB)
- Physical state: Clear colourless liquid but solidified to an opaque white crystalline solid after receipt
- Analytical purity: Diethylene glycol dibenzoate - 97.67% (area %)
- Impurities (identity and concentrations):
Dipropylene glycol dibenzoate - 1.34% (area %)
Ethylene glycol dibenzoate - 0.15% (area %)
Propylene glycol dibenzoate - 0.11% (area %)
n-Propyl benzoate - 0.054% (area %)
Diethylene glycol monobenzoate - 0.047% (area %)
Unknown #1 - 0.19% (area %)
- Lot/batch No.: 558260E1
- Expiration date of the lot/batch: Not stated
- Storage condition of test material: Room temperature, dessicated

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) 5 wks; (F1) 6 wks
- Weight at study initiation: (P) Males 207 ± 22.0, 203 ± 20.6, 204 ± 15.8 and 198 ± 19.4 g (Groups 1 to 4 respectively) and for the females 160 ± 13.5, 161 ± 14.9, 159 ± 10.3 and 156 ± 13.7 g (Groups 1 to 4 respectively)
- Housing: Stainless steel or high density polypropylene 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: public 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 8 days in advance of use.
- 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 20 days, were confirmed for DEGDB in LAD 2 at nominal concentrations of 500 ppm and 20000 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 20 days were confirmed for DEGDB in LAD 2 formulation at nominal concentrations of 500 ppm and 20000 ppm (Huntingdon Life Sciences Report VCL321/990090).
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 17)
- Pairing for second generation (week 28)
- Lactation for second generation (week 34)


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 5% above and 5.2% 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 11 weeks before pairing and until termination when litters were weaned.
Frequency of treatment:
Treated feed was available ad libitum.
Details on study schedule:
- F1 parental animals were not mated until 11 weeks after selected from the F1 litters.
- Age at mating of the mated animals in the study: P (F0) 16 weeks, (F1) 15-16 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
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. VCL321/990090).
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. P (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 females was recorded for Days 1-6, 7-13, 14-17 and 18-20 of lactation
Food consumption for F1 females was recorded on 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 29 days of pairing of 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 spermatids
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).
Other offspring that died before scheduled termination were subject to external and internal examination F1 offspring following weaning not selected for continuation of the study were killed around Day 31 of age all pups selected for organ weights were killed on Day 31 following the selection process for the next generation F2 offspring were killed on Day 21 of age.

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 WEIGTHS
Organs were taken from 1 male and 1 female randomly selected from each litter on Day 31 for F1 offspring and Day 21 for F2 offspring 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 of females during gestation and lactation, litter data including offspring bodyweights, sexual development data and organ weights).
- 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 wasused.
- Dependent on the heterogeneity of variance between treatment groups parametric tests analysis of variance, followed by Williams' test or non parametric tests (Kruskal Wallis) followed by Shirley's test were used as appropriate (bodyweight and food consumption data during gestation and lactation, litter data, sexual
development data, seminology data and offspring organ weights).
- Where 57% or more of the values for a given variable were the same 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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

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.
Amongst females there were four unscheduled deaths one at each treatment level including Control. These deaths appear to be related to coincidental problems associated with parturition and neither the incidence nor distribution indicated an association with treatment.

F1
The general condition of F 1 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: Overall there was no conclusive effect of treatment with DEGDB on bodyweights or bodyweight gain of the F0 males at any inclusion level bodyweight gain of treated males at termination after about 16 weeks of treatment was essentially similar to Controls.

Females: There was no obvious adverse or significant effect of treatment with DEGDB on bodyweights or bodyweight gain of the F0 females during the 10 week pre pairing treatment phase at any inclusion level or during gestation or during lactation period.

F1
Males: There was no effect of treatment with DEGDB5 on bodyweight gain of the F1 males to termination at any inclusion level.
Females: Mean bodyweight at the formal start of the Fl generation approximately 4 weeks of age were essentially similar in all groups. At 10000 ppm there was a suggestion of lower bodyweight gain during the pre pairing treatment period. There was no effect of treatment with DEGDB5 on bodyweight gain during the pre pairing treatment period at 1000/3300 ppm.
Bodyweight gains of females during gestation were not obviously affected by treatment at any inclusion level. Following parturition bodyweight change between Day 0 of gestation and Day 1 of lactation for those dams that successfully reared their offspring to weaning did not indicate any adverse effect of treatment on the bodyweight of the parental females.
At 10000 ppm mean bodyweight loss was evident during the first 4 days of lactation and bodyweight gain was also marginally lower at 3300 ppm compared to the Control differences however failed to attain statistical significance.
There was no obvious adverse effect of treatment on food consumption of either F1 males or females during the 10 week pre pairing treatment period.


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 7-13 the intake of the females was in excess of 2000 mg/kg/day at 10000 ppm.

F1
Exposure levels well in excess of 500 mg/kg/day were achieved at 10000 ppm throughout the 11 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 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 DEGDB on oestrous cycles at any dietary inclusion level the majority of F0 females in each group showed a regular 4 or 5 day cycle.

F1
There was no adverse effect of treatment with DEGDB on oestrous cycles at any dietary inclusion level the majority of F1 females in each group showed a regular 4 or 5 day cycle.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
F0
There was no adverse effect of exposure to DEGDB on the male reproductive system as assessed by sperm motility progressive sperm motility sperm count homogenisation resistant spermatids and sperm morphology.

F1
There was no adverse effect of exposure to DEGDB on the male reproductive system as assessed by sperm motility progressive sperm motility sperm count homogenisation resistant spermatids and sperm morphology.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
F0
Precoital interval and mating performance
The majority of F0 animals in all groups showed a positive indication of mating within the first four days of pairing. The incidence and distribution of animals failing to mate within these four days did not indicate any adverse effect of treatment.
All F0 animals mated and with the exception of three females (1 at 1000 ppm and 2 at 10000 ppm) all matings lead to pregnancy in the female partner.
While pregnancy rate was lowest at the highest dosage fertility was still very high and in the absence of a similar finding in the F1 generation where
animals were exposed to the test material for a longer period was considered unaffected by treatment at this inclusion level.

Gestation length gestation index and parturition
The distribution of gestation lengths and gestation index were essentially similar in all groups. There was no evidence that treatment with DEGDB had any influence on the parturition process.

F1
Pre coital interval and mating performance
The majority of F1 animals in all groups showed a positive indication of mating within the first four days of pairing. The incidence and distribution of animals failing to mate within these four days did not indicate any adverse effect of treatment.
The pregnancy rate was generally good for all groups.

Gestation length gestation index and parturition
The distribution of gestation lengths and gestation index were essentially similar in all groups. There was no evidence that treatment with DEGDB had any influence on the parturition process.


ORGAN WEIGHTS (PARENTAL ANIMALS)
F0
Intergroup differences in absolute and bodyweight relative organ weights for the F0 parental animals did not indicated any adverse effect oftreatment with DEGDB in either sex.

F1
Inter group differences in absolute and bodyweight relative organ weights for the F0 parental animals did not indicated any adverse effect of treatment with Benzoflex 2-45 in either sex.


GROSS PATHOLOGY (PARENTAL ANIMALS)
F0
Neither the incidence type nor distribution of the findings observed during the necropsy of F0 parental animals indicated any adverse effect of treatment with Benzoflex 2-45.

F1
Neither the incidence type nor distribution of the findings observed during the necropsy of F1 parental animals indicated any adverse effect of treatment with Benzoflex 2-45.


HISTOPATHOLOGY (PARENTAL ANIMALS)
F0
Microscopic examination of the organs and tissues taken from 10 F0 males and 10 F0 females from the control and 10000 ppm groups did not reveal any findings that were considered to be related to exposure to DEGDB. Additionally all abnormalities observed at necropsy were examined microscopically for all animals and did not indicate any adverse effect of treatment.

Oestrous cycle at termination (parental F0 animals)
Vaginal smears were assessed for parental females for approximately one week after weaning (Days 22 to 28 of lactation). With the exception of one female receiving 1000 ppm all surviving females were confirmed to have returned to oestrus cycling after lactation.

F1
Microscopic examination of the organs and tissues taken from 10 F0 males and 10 F0 females from the control and 10000 ppm groups did not reveal any findings that were considered to be related to exposure to DEGDB.
Additionally all abnormalities observed at necropsy were examined microscopically for all animals and did not indicate any adverse effect of treatment.

Oestrous cycle at termination (parental F1 animals)
Vaginal smears were assessed for parental females for approximately one week after weaning (Days 22 to 28 of lactation). All surviving females were confirmed to have returned to oestrus cycling after lactation.

Ovarian primordial follicle counts F1 females
Ovarian primordial follicle counts were conducted on 10 Control F1 females and 10 F1 females at 10000 ppm and in addition any decedent female F1 animals. There was no obvious adverse effect of treatment with Benzoflex 2-45 onthe primordial follicle populations.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOEL
Remarks:
equivalent to a minimum estimated daily achieved dosage of 500 mg/Kg/d
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
F1
There was no obvious adverse effect of treatment on litter size or pup survival as assessed by the number of uterine implantation sites recorded at termination litter size at Day 1 survival of offspring to litter standardisation on Day 4 and subsequent survival to weaning.

F2
Although the incidence of total litter loss was high, no association with treatment was considered proven. There was no obvious adverse effect of treatment on litter size or pup survival as assessed by the number of uterine implantation sites recorded at termination litter size at Day 1 survival of offspring to litter standardisation on Day 4 and subsequent survival to weaning.


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 bodyweight at Day 1 was lower than Control for all treatment groups differences being most noticeable at 1000 and 3300 ppm. At these lower inclusion levels the differences in mean pup weight was considered to principally reflect the slightly larger mean litter size observed.
At 10000 ppm mean pup weight was only slightly lower than Control however litter size was also slightly lower so offspring bodyweight might have been expected to be more closely matched to Control values.
While this may be suggestive of a threshold effect in view of the small differences observed an effect of treatment was not considered proven.
There was no clear effect on subsequent bodyweight gain to weaning for offspring of either sex at any of the inclusion levels investigated.

F2
At 10000 ppm bodyweights of F2 offspring of either sex at Day 1 was lower than Control. While to some extent this may reflect the slightly larger litter size at this dosage in view of the similar observation for the F1 offspring, this may be related to treatment. Subsequent bodyweight gain to weaning for both sexes were lower than Control differences for male offspring attaining statistical significance.

At lower dosages it was not considered that offspring bodyweight on Day 1 and subsequent gain to weaning had been adversely affected by treatment.


SEXUAL MATURATION (OFFSPRING)
F1
Assessment of the sex ratio on Day 1 did not indicate any selective effect of treatment on post-implantation survival for either sex.
Similarly sex ratio from Day 1 to litter standardisation on Day 4 of age and after that to weaning did not indicate any apparent effects of treatment upon the survival of either sex during the post natal and pre weaning periods.

Sexual development as assessed by age and bodyweight at the time of the attainment of vaginal opening in F 1 females and balano preputial separation in F1 males was unaffected by treatment with DEGDB.

F2
Assessment of the sex ratio on Day 1 did not indicate any selective effect of treatment on post implantation survival for either sex. Similarly sex ratio from Day 1 to litter standardisation on Day 4 of age and later to weaning did not indicate any apparent effects of treatment upon the survival of either sex during the post natal and pre weaning periods.


ORGAN WEIGHTS (OFFSPRING)
F1
Brain spleen or thymus weights were recorded for the unselected F1 offspring and comparison of mean absolute and bodyweight relative values did not indicate any adverse effect of exposure to DEGDB.

F2
At day 21, at 10000 ppm absolute and bodyweight relative spleen weights ofboth sexes of the F2 offspring were lower than Control; differences being most noticeable and attaining statistical significance amongst females.
Absolute and bodyweight relative values for the brain and thymus at this dosage were comparable to Control.
At 1000 and 3300 ppm intergroup differences in absolute and bodyweight relative brain spleen and thymus weights of F2 offspring did not indicate any adverse effect of exposure to DEGDB.


GROSS PATHOLOGY (OFFSPRING)
F1
Macroscopic examination of offspring dying before weaning did not reveal any findings considered to be related to treatment with DEGDB. Deaths generally occurred by Day 4 of age and 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 DEGDB. Deaths generally occurred by Day 4 of age and 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.


HISTOPATHOLOGY (OFFSPRING)
F1
Neither the incidence type nor distribution ofthe findings observed indicated any adverse effect of treatment.

F2
Neither the incidence type nor distribution of the findings observed indicated any adverse effect of treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on lack of adverse treatment-related effects observed at the highest concentration tested.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

Effect levels (F2)

Key result
Dose descriptor:
NOEL
Generation:
F2
Effect level:
3 300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slight body weight reduction in F2 offspring.

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The evidence from this study suggested that a dietary concentration of diethylene glycol dibenzoate at 10000 ppm should be considered as the No Observed Adverse Effect Level (NOAEL) for P (F0) and F1 parent animals. The NOAEL for the developing offspring is considered to be 3300 ppm. The No Observed Effect Level (NOEL) for reproductive parameters is considered to be 10000 ppm.
Executive summary:

A two generation study in Sprague-Dawley rats was conducted to assess the effects on reproductive performance of the test material DEGDB (HLS 2001, VCL 322/003021). The study was conducted according to OECD and EPA test guidelines, and in compliance with GLP.

Dietary administration of DEGDB 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 consistent with expectations 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. There were no obvious toxicological effects of treatment for the two generations on the general condition of the parental animals, although a slight decrease of the maternal weight change was noted at 10000 ppm in both generations and at 3300 ppm in the F1 generation. There was no effect on fertility and reproductive performance at any of the dietary exposure levels in either generation. Litter parameters at birth of the F1 and F2 progeny and their survival to weaning showed no apparent detrimental effects of treatment with DEGDB. However, there was a reduction in weight gain from birth to weaning in the F2 offspring at 10000 ppm. No abnormal findings were apparent at necropsy of the F0 or F1 parental animals, the post-weaned unselected F1 offspring, or the F2 offspring. Organ weight assessment of the F0 and F1 parent animals did not suggest any adverse effects of DEGDB on any organs. Assessment of spermatogenesis and histopathology in both parental generations showed that there were no injurious effects on the testes or other reproductive organs. Furthermore, detailed histopathological examination of the tissues from both sexes in both generations did not reveal any adverse effects of treatment with DEGDB. The only possible effect of DEGDB treatment detected at assessment of organ weights from F1 and F2 offspring was a statistically significant lower absolute and relative (to bw) spleen weights among F2 males and females compared with Controls.

The evidence from this study suggested that a dietary concentration of DEGDB 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 (equivalent to a minimum estimated daily achieved dosage of 500mg/kg/d).