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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 24,1997 to 29 January 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted according to recognised test methods. For read-across justification see Section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: BISOFLEX L911P
- Formal name: 1,2 benzene dicarboxylic acid , di C9-C11 alkyl esters
- Physical state:liquid oily
- Analytical purity:99.2%
- Lot/batch No.:PLA/S20517/97
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles river UK Limited
- Age at study initiation:5 weeks
- Weight at study initiation: 145 - 228 g (males) , 115-198 g (female)
- Fasting period before study:
- Housing: TR18 cages or RB3 from arrowmight biosciences ; stainless steel
- Diet:ad libitum
- Animal diet quality: LAD 2 SQC manifactured by special diet services limited. This powdered diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum
- Water quality: is governed by regulation published by department of the environment. At approximately six-monthly intervals , water was routinely sampled for analysis by a laboratory independent of the supplier, for selected chlorinated pesticides and polychlorinated biphenyl contaminants.
- Acclimation period:5 days
- Healty check: during the acclimatation period
- identification: by tail tattoo
ENVIRONMENTAL CONDITIONS
- Temperature (°C):21°C
- Humidity (%):42-71%
- Air changes:filterd fresh air
- Photoperiod : 12 hours cycle dark/light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet: The required dietary concentrations were prepared by direct dilution of the premix with further quantities of untreated diet. Blending was achieved by mixing in a Turbula Mixer for a minimum period of 6 minutes (for a least 100 cycles).
- Mixing appropriate amounts with: A pre-mix of diet was prepared by weighing out the quantity of D911P and adding an approximately equal amount of untreated diet. This mixture was then stirred prior to the addition of a further quantity of diet, approximately equal to the weight of the mixture; this process was repeated until the mixture did not readily cohere. The admixture was then passed through a 1 mm sieve. Further quantities of untreated diet were added until the required quantity of premix was obtained, the premix was then stirred thoroughly. Blending was achieved by mixing in Turbula Mixer for a minimum period of 6 minutes (for at least 100 cycles)

VEHICLE
- Justification for use and choice of vehicle:to simulate the conditions of human exposure
- Concentration in vehicle:0 ppm (control) , 1000 ppm , 5000 ppm , 10000 ppm , 20000 ppm( was selected as the initial concentration for the highest dosage gruop, but this was reduced to 10000 ppm after 6 weeks of treatment [43 days] of F0 generation , following poor bodyweight gain by males)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating
- Proof of pregnancy: Evidence of vaginal plug and sperm in vaginal smear. Referred to as Day 0 of gestation
- After 4 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: Not applicable
- After successful mating each pregnant female was caged: Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Specified formulations prepared before the preliminary study were analysed to assess the homogeneity and stability of the test substance in the diet matrix at concentrations of 100 and 20000 ppm. The results indicated that the mixing procedure generated homogenous distributions of the test material in powdered diet and that the material was stable in the diet under conditions of use. No further homogeneity or stability analyses were performed.
Single samples (nominally 200g) were taken from diet samples (2 assays from each sample) to check for the content of the compound at weeks 1, 8 11 14, 18, 28; and 31 of the study The results of the test diet formulations analysed during the study were within 12% of the nominal concentrations
confirming the accuracy of the formulation.
The samples were analysed using methanol extraction from the diet, methanol dilution and iniection of the diluited test samples on to a high-performance liquid chromatograph (HPLC) with ultra- violet detection. The amount of D911P in the test samples was quantified by reference to calibration standards of low concentrations.
Duration of treatment / exposure:
The substance was administered continuously in the diet at concentrations of 1000, 5000 or 10000 ppm to groups of rats throughout the two generations. The highest dosage group animals were given 20000 ppm of D911P for approximately six weeks (43 days) at the start of the Fo generation but this was reduced to 10000 ppm because of a marked reduction in bodyweight gain of the males. A fourth group received the basal diet without the test material and served as the Control.
The Fo generation received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation. From the F1 litters, 28 male and 28 female offspring were selected to form the F1 generation. Both sexes received treatment for a minimum of 10 weeks from the formal commencement of the F1 generation (approximately 28 days of age), throughout pairing, gestation and lactation.
Frequency of treatment:
Continuous, via the diet.
Details on study schedule:
- F1 parental animals not mated until 12-13 weeks after selection from the F1 litters.
- Selection of parents from F1 generation when pups were 2-3 weeks days of age.
- Age at mating of the mated animals in the study: 15 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
Reduced from intial concentration of 20000 ppm
No. of animals per sex per dose:
F0 generation - 28 males/28 females per group
F1 generation - 28 males/28 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on results of a preliminary study. Highest dose selected was maximum recommended by test guidelines
- Rationale for animal assignment (if not random): Random
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations included any visible signs of reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly; females - weekly, Days 0, 6, 13 and 20 of gestation and Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

Oestrous cyclicity (parental animals):
Examined for 10 days before pairing and until evidence of mating
Sperm parameters (parental animals):
The quality of epididymal sperm and the numbers of homogenisation resistant spermatids was unaffected by treatment in either the Fo or the F1 generation males.

Parameters examined in all/P/F1 male parental generations:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, homogenisation-resistant spermatids.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
For external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
Findings in the Fo and F1 generation considered to be related to treatment with D911P were generally restricted to the livers of males treated at 10000 ppm. The range of findings observed for the high dosage group males is indicative of hepatotoxicity of the periacinar hepatocytes with consequential increased cell turnover resulting in regenerative hyperplasia. The development of altered cell foci in males at this dosage is probably a consequence of the increase turnover of hepatocytes, while bile duct proliferation is probably associated with the altered architecture of the liver.
SACRIFICE
- Male animals: All surviving animals killed as soon as possible after the last litters in each generation were weaned
- Maternal animals: All surviving animals were killed after the last litter of each generation was weaned.

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

ORGAN WEIGHTS
Parental animals - Adrenal glands, brain, epididymides (L&R), kidneys, liver, ovaries (L&R), prostate, seminal vescicles and coagulation gland, spleen, testes (L&R), thymus and uterus with cervix
Unselected F1 and F2 - Brain, liver, spleen thymus

HISTOPATHOLOGY
Parental animals - Abnormalities, epididymides (R), liver, mammary glands (caudal), ovaries, oviduct, pituitary, prostate, seminal vescicles and coagulation gland, testis (R), thymus, uterus, cervix and vagina
Unselected F1 and F2 - Abnormalities
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 25 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

ORGAN WEIGHTS
Brain, liver, spleen thymus

HISTOPATHOLOGY
Macroscopic abnormalities
Statistics:
Significance tests , employing of variance followed by an inter-group comparison with the control
Reproductive indices:
Pre-coital interval, duration of gestation, maternal behaviour, number of live/dead offspring, pup body weight, sex ratio
Offspring viability indices:
Number live/dead pups, body weight,

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
bodyweight decrease
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
bodyweight decrease
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
male liver
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
increased cell turnover
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)

F0 generation
Clinical signs and mortality
The general condition of animals was similar in all groups. Observed signs were largely restricted to areas of hair loss, coat staining and, to a lesser extent, scabbing.
There were three mortalities in the F0 generation. One Control male was killed during Week 6 due to a persistent ulceration on its upper dorsal surface. One female receiving 5000 ppm was killed after showing hunched posture, piloerection, thin appearance and apparent blood and urine staining on the ventral body surface on Day 5 of lactation (Week 15 of treatment), Bodyweight on Day 4 of the majority of the ljtter was lower than that recorded on Day 1 and the litter was showing signs of neglect (pups cold, unfed and underactive) on Days 4 and 5 of lactation. Necropsy failed to reveal an obvious cause for the animals’ condition. One female receiving 10000/20000 ppm was killed during Week 16 after showing apparent bleeding from the vagina 2 days after parturition. Other clinical signs included hunched posture, piloerection, cold to touch, pallor and underactivity. Necropsy findings were unremarkable. The incidence and circumstances of the deaths of the two treated females did not indicate any association with treatment with the substance

Bodyweight
Males - At 20000 ppm mean bodyweight gain of males was significantly lower than the concurrent Control; the divergence being such that it was considered necessary to reduce the dietary exposure for this group during the seventh week of treatment to 10000 ppm, in order to safeguard the reproductive objectives of the study. Subsequent weight gain stabilised; however, the deficit in bodyweight gain was never recovered and therefore the mean values for overall gain and absolute bodyweight of these animals at termination was significantly lower than the Control.
Females - At 20000 ppm mean bodyweight gain of females was lower than the concurrent Control and, although this effect of treatment was not as marked as observed for the males, differences had attained statistical significance by the end of the third week of treatment. With the reduction in dietary exposure to 10000 ppm during the seventh week of treatment subsequent weight gain to the end of the pre-pairing period was essentially similar to the Control; as observed with the males, however, the deficit in bodyweight gain was never recovered and therefore the mean values for overall gain and absolute bodyweight of these animals at the end of the pre-pairing period remained significantly lower than the Control. At 5000 ppm initial mean bodyweight gain of females was similar to that of the Control. As the pre-mating period progressed some divergence was apparent, although differences never attained statistical significance. There was no obvious adverse effect of treatment at 1000 ppm on bodyweight gain of females prior to pairing. Overall bodyweight gain during gestation was similar in all groups and was not influenced by treatment. Lower bodyweight gain was noted in treated groups during the first week of gestation; however this was considered likely to be a reflection of the general pattern established in the week preceding pairing and the slightly lower bodyweight at 5000 and 10000/20000 ppm. In the absence of any effects on overall bodyweight gain during gestation, or effects on subsequent fertility, litter data or pup weights at Day 1 this finding is not considered to be of any toxicological significance.
The pattern of bodyweight change during lactation was similar in all groups with the exception that at 10000 ppm the drop in bodyweight frequently observed during the last week of lactation did not materialise. Again, the significance of this finding is unclear, as the bodyweight gain of F1 offspring during this period appeared unaffected, but a similar response during lactation was observed at this dosage in the F1 generation; nevertheless, it is not considered to represent an adverse effect of treatment.

Food consumption
Males - Food consumption amongst males receiving 10000/20000 was notably lower than that of Controls throughout the pre-pairing period, these differences attaining statistical significance for nine of the ten weeks. Food consumption of males fed a diet containing 5000 or 1000 ppm was generally comparable with that of the control and appeared unaffected by the presence of the substance in the diet.
Females - The pattern of food consumption for females during the pre-pairing period or during gestation and lactation did not indicate that food intake had been affected by the presence of the substance in the diet, even during the first 6 weeks when the highest inclusion level was 20000 ppm.

Food conversion efficiency
Males - Food conversion efficiency of males receiving 20000 ppm was notably lower than Controls until the dietary concentration was lowered to 10000 ppm during Week 7. While food conversion for these animals initially improved there was a suggestion that efficiency declined again towards the end of the pre-mating period. There were no consistent effects of treatment upon food conversion efficiency at 1000 or 5000 ppm.
Females - Food conversion efficiency of females receiving 20000 ppm appeared to show some impairment during Weeks 5 and 6 of treatment; prior to this it had been comparable to Controls. Following the lowering of the dietary concentration to 10000 ppm during Week 7 food conversion was generally comparable to Control for the remaining pre-mating period. There were no consistent effects of treatment upon food conversion efficiency at 1000 or 5000 ppm.

Achieved chemical intake
For animals receiving 20000 ppm until Week 7 chemical intake for males ranged from 2379 mg/kg/day in Week 1 to 1366 mg/kg/day in Week 6 and for females ranged from 2403 to 1610 mg/kg/day over the same period. Following the reduction in dietary inclusion to 10000 ppm chemical intake during the remaining pre-pairing period fell by approximately half, then continued to show the natural decline associated with change in growth rate: food intake as animal’s mature; intake immediately prior to pairing was 619 mg/kg/day in males and 696 mg/kg/day in females.
Chemical intake during the pre-pairing period by animals in the lower treatment groups was in proportion to the dietary concentrations, reducing steadily as the animals became older. At 5000 ppm the achieved ranges for males were 607 mg/kg/day in Week 1 to 280 mg/kg/day in Week 10 and 606 to 328 mg/kg/day for females over the same period. At 1000 ppm the achieved ranges over the same period were 121 to 56 mg/kg/day for males and 123 to 63 mg/kg/day for females. Chemical intake by females during gestation was slightly higher than in the preceding period before pairing and approximately doubled during mid lactation in line with the increase in food intake at peak physiological demand.

Oestrous cycles
There was no obvious adverse effect of treatment on the regularity and duration of the oestrous cycles at any of the dietary inclusion levels investigated.

Pre-coital interval and mating performance
There was no adverse effect of treatment on pre-coital interval, the majority of animals mating within four days of pairing i.e. at the first oestrus opportunity. Fertility was not affected by treatment at any of the dosages investigated.

Gestation length and parturition
The majority of females gave birth to live young after gestation periods of between 22 and 23.5 days. Within this range there appeared to be a slight shift in treated animals towards having a slightly shorter gestation period, the trend attaining statistical significance. The biological significance of this observation in this study is uncertain as there appeared to be no adverse effect on survival at parturition or on bodyweight at Day 1, however it was replicated in the F1 generation and may have been linked to the lower bodyweights of females at 5000 and 10000 ppm.
There were no indications of any adverse effects of treatment upon the parturition process.

Litter size and offspring viability
The number of implantations sites recorded at necropsy were similar for all groups and there was no obvious adverse effect of treatment on in-utero survival, as assessed by total litter size and live litter size at Day 1 of age, or subsequent pup survival to weaning. Indices of offspring viability were largely influenced by the incidence of total litter losses (2, 3, 3 and 2 litters for Control, low- mid- and high-dose groups), in addition to the death of a dam at 5000 and 10000 ppm. The majority of litter deaths occurred during the first week of age with few deaths after litter size standardisation at Day 4 and, in the majority of cases, litters that died were small in bodyweight when first seen and were considered to be cold, unfed and underactive before eventual death.

Sex ratio
Sex ratio at birth was not influenced by treatment and there was no selective effect on pup survival of either sex.

Sperm evaluation of F0 males
The quality of the epididymal sperm, as assessed by numbers, motility and gross morphology was unaffected by treatment at any of the inclusion levels investigated and there was no evidence of an effect of treatment upon the homogemsation resistant spermatids within the testis.
It was noted that three males (one in the control group, one in the low-dose group and one in the mid-dose group) were siblings and all three showed a low percentage of progressively motile sperm and failed to induce pregnancy in their female partners.

Necropsy of adults
Macroscopic necropsy findings considered to be related to treatment were predominantly restricted to liver changes amongst males receiving 10000/20000 ppm. The majority of livers typically were pale and showed areas of macroscopic change, some also having an irregular surface. For females at this level a few animals showed enlargement of the liver and accentuation of the lobular pattern however, the findings common to their male counterparts were generally not evident.
For males at 1000 and 5000 ppm liver changes were generally restricted to swelling, with only the occasional animal at 5000 ppm showing the changes typical of the high dosage. For females at 1000 and 5000 ppm liver findings were generally unremarkable. Neither the type, incidence or distribution of findings for other organs and tissues at necropsy indicated any adverse effects of treatment at any of the inclusion levels investigated.

Organ weights of adults
At 10000/20000 ppm the mean bodyweight of males at termination was significantly lower than that of the Control complicating the assessment of possible effects of treatment on organ weights. For males at 1000 and 5000 ppm and females at all dosages terminal bodyweight was comparable to the control.
For males at 10000/20000 ppm a number of absolute organ weights (brain, liver, kidneys, spleen, adrenals, epididymides, prostate and seminal vesicles) and bodyweight relative organ weights (brain, kidneys, spleen, thymus, testes, epididymides and seminal vesicles) were statistically significant, when compared with the concurrent Control. In general bodyweight relative organ weights were considered to give a better indication of effects of treatment than absolutes at this inclusion level because of the marked effect on bodyweight. The most notable differences from Control were considered to be increases in the relative weight of the thymus, spleen and kidneys; however in the absence of any histopathological changes the toxicological significance of these findings is unclear. For males at 5000 and 1000 ppm there were no obvious adverse effects of treatment on organ weights.
Amongst females, increased liver weight at 5000 and 10000/20000 ppm was considered to be the most noticeable effect, particularly as it suggested a different response from the males. Decreased absolute and relative adrenal, thymus and, to a lesser extent, spleen weights at 10000/20000 ppm may also be of some toxicological significance but, as for males, in the absence of any histological changes this is not clear. Excluding the increased liver weight at 5000 ppm organ weights at this dosage and at 1000 ppm were not obviously affected by treatment.

Histopathology of adults
Histopathological findings in the F0 generation considered to be related to treatment were restricted to changes in the livers of males at 5000 ppm and of both sexes at 10000/20000 ppm. The range of findings observed for the high-dose group males is indicative of hepatotoxicity of the periacinar hepatocytes with consequential increased cell turnover resulting in regenerative hyperplasia. The development of altered cell foci and the single occurrence of a hepatocellular adenoma in males at this dosage is probably a consequence of the increase turnover of hepatocytes, while bile duct proliferation is probably associated with the altered architecture of the liver. Changes in the liver may be associated with an increase in peroxisomal enzymes which has been demonstrated in the livers of five treated F1 animals at this dosage. In contrast to the males, treated females were markedly less affected with only a low incidence of periacinar hepatocytic vacuolation, periacinar hepatocytic hypertrophy and periacinar hepatocytic necrosis being observed at the high dosage group.
There were no other microscopic findings observed that were considered to be related to treatment.
It was noted that females receiving 10000/20000 ppm had a lower incidence of dilated uteri and a lower incidence of uteri with dilated endometrial glands. Dilatation of the uterus reflects the normal physiological response of the uterus to the hormonal status of the animal during the oestrous cycle. As these animals were still in the immediate post lactation phase at the time of necropsy, it is unlikely that the oestrous cycle was fully established or regular in many of the animals. The lower incidence of dilated uteri at the highest dosage is considered to be fortuitous and of no toxicological significance.

F1 generation
Clinical signs and mortality
The general condition of animals was similar in all groups and clinical signs were largely restricted to areas of hair loss, coat staining and, to a lesser extent, scabbing.
There were seven mortalities in the F1 generation. One male treated at 10000 ppm was found dead in Week 13, necropsy revealed the anterior lateral lobe of the liver to be swollen. Given the isolated nature of this death it is considered most likely to be coincidental. The other mortalities in this generation concerned females either at or within a few days of parturition; given the group distribution (2, 2, 2 and 0 in Control, low-, mid- and high-dose groups respectively) it is considered that these deaths were due to coincidental factors rather than any adverse influence of treatment.

Bodyweight
Males - At 10000 ppm initial bodyweight of males at the formal start of the F1 generation was within 97% of the control value. Initially, weight gain was fairly similar to that of the Control but subsequent gain was slightly lower, with differences from Control attaining statistical significance from Week 5 of the generation (around 9 weeks of age). At pairing bodyweight gain was 89% of that achieved by the control group and was 87% of the Control at termination about seven weeks later; at this point the animals had had direct exposure to the test material for about 20 weeks.
Bodyweight and bodyweight gain of males receiving 1000 and 5000 ppm were similar to control values throughout the generation.

Females - Initial bodyweights of females at the formal start of the F1 generation was similar in all treated groups and within 95% of the control value. Bodyweight gain of females at all inclusion levels were generally comparable to the Control throughout the pre-pairing period; however because gain at 10000 ppm was slightly lower than Control towards the end of the period overall gain was slightly lower than Control.
Overall bodyweight gain during gestation was lower in all treated groups compared to the Control. At 5000 and 10000 ppm this was principally due to lower bodyweight gain during the first two weeks of gestation; differences from Control attaining statistical significance but not showing a consistent dosage-relationship. This was considered to reflect the general pattern of bodyweight observed in the pre-pairing period. At 1000 ppm bodyweight gain during the first two weeks of gestation was essentially similar to the control and the subsequent slight divergence from Control is considered to reflect the slightly higher litter size in the control and is not considered to be of any toxicological significance.
The pattern of bodyweight change during lactation was similar in all groups with the exception that at 10000 ppm the drop in bodyweight that often occurs during the last week of lactation was not seen at this inclusion level. While this reflected a pattern already observed at this dosage in the F0 generation it should be noted that actual gain during lactation prior to this had been notably lower than observed in other groups. In addition, unlike the F0 generation an effect on bodyweight gain of the offspring (F2) was apparent during this period and differences in physiological demand by the litter would have had an influence on maternal bodyweight.

Food consumption
Males - Food consumption amongst treated males was unaffected by the presence of the substance in the diet, at all inclusion levels, during the pre-pairing period, being essentially similar or superior to that of the controls.
Females - Food consumption amongst treated females was unaffected by the presence of the substance in the diet, at all inclusion levels, during the pre-pairing period or during gestation and lactation.

Food conversion efficiency of males and females before pairing
While there was no clear effect of treatment on the efficiency of food utilisation for either sex at any inclusion level throughout the pre-pairing period, there was a suggestion of impaired efficiency at 10000 ppm towards the end of this phase.

Achieved chemical intake
As anticipated chemical intake by animals was in proportion to the dietary concentrations, reducing steadily as the animals became older. Chemical intake of females during gestation was slightly higher than it had been immediately before pairing and approximately doubled during mid lactation in line with the increase in food intake at peak physiological demand.

Sexual maturation
There was no obvious adverse effect of treatment on sexual maturation in the F1 generation, as assessed by the mean age of attainment of vaginal opening in females and balano-preputial separation in males, at any of the dietary inclusion levels investigated.

Oestrous cycles
There was no obvious adverse effect on the regularity and duration of the oestrous cycles at any of the dietary inclusion levels investigated.

Precoital interval and mating performance
There was no adverse effect of treatment on pre-coital interval, the majority of animals mating within four days of pairing i.e. at the first oestrus opportunity. Fertility was not considered to have been affected by treatment at any of the dosages investigated.

Gestation length and parturition
The majority of females gave birth to live young after gestation periods of between 22 and 23 days. Within this range there appeared to be a marginal shift in animals receiving 5000 and 10000 ppm towards having a slightly shorter gestation period, the trend attaining statistical significance at the highest dosage. This trend was also seen in treated groups in the F0 generation. The biological significance of this observation in this study is uncertain but may have been linked to the lower bodyweight of females at these inclusive levels.

One female receiving 10000 ppm was not observed to have given birth to live young but was found at necropsy to have a single implantation site. The loss of a litter comprising of a single pup is not particularly unusual and, in the absence of any evidence of an effect on implantation loss in other animals at this dosage, this finding is considered to be coincidental and unrelated to treatment. Two animals receiving 1000 ppm and one animal receiving 5000 ppm were killed or died during parturition. In addition one control animal and one animal receiving 5000 ppm were killed in early lactation due to their poor condition. Given the absence of any treatment related pattern, these events are considered to be coincidental.

Litter size and offspring viability
The number of implantation sites recorded at necropsy was similar for all groups and there was no obvious adverse effect of treatment on in-utero survival, as assessed by total litter size and live litter size at Day 1 of age, or subsequent pup survival to weaning. Indices of offspring viability were largely influenced by the previously discussed parental mortalities rather than total litter loss, which were restricted to 0, 2, 1 and 0 occurrences for Control, low-, mid- and high-dose groups respectively.

Sex ratio
Sex ratio at birth was not influenced by treatment and there was no selective effect on pup survival of either sex.

Sperm evaluation of F1 males
The quality of the epididymal sperm, as assessed by numbers, motility and gross morphology was unaffected by treatment at any of the inclusion levels investigated and there was no evidence of an effect of treatment upon the homogenisation resistant spermatids within the testis. One Control animal had agenesis of the left testis and was not assessed. One animal receiving 10000 ppm had reduced and flaccid epididymides and testes; sperm count, motility and morphology assessment from an epididymal sample was not possible. This animal failed to induce pregnancy in its female partner.

Necropsy of adults
Macroscopic necropsy findings considered to be related to treatment were predominantly restricted to liver changes amongst males receiving 10000 ppm. The majority of livers were pale and there was a significant incidence of accentuated lobular pattern, irregular surface and, to a lesser extent, small appearance. For females at this dosage a number of animals showed enlargement, accentuated lobular pattern or swollen liver and there was also a increased incidence of animals with a small thymus. For both sexes at 5000 ppm liver changes were restricted to a low incidence of swollen appearance, accentuated lobular pattern and, for males only, pallor in a few animals. For both sexes at 1000 ppm liver findings were unremarkable. Neither the type, incidence or distribution of findings for other organs and tissues at necropsy indicated any adverse effects of treatment at any of the inclusion levels investigated.

Organ weights of adults
At 10000 ppm the mean bodyweight of males, and to a lesser extent females, at termination was significantly lower than that of the Control. Differences however were not as marked as those observed for the high dosage males in the Fo generation. For both sexes at 1000 and 5000 ppm terminal bodyweight was comparable to the control. For males at 10000 ppm a number of absolute organ weights (liver, kidneys, spleen, adrenals, epididymides and seminal vesicles) were statistically significant, when compared with the concurrent Control. Due to the lower bodyweight at this dosage bodyweight-relative organ weights were generally considered to give a better indication of effects of treatment than absolutes and most of the organ weight differences noted in males disappeared when the values were considered as a percentage of actual bodyweight. Organ weights of males, taking into consideration the effect on bodyweight at the highest dosage, were not considered to have been adversely affected by treatment. For females at 10000 ppm a number of absolute organ weights (liver, spleen, adrenals, thymus and ovaries) and relative organ weights (brain, liver, kidneys, spleen, adrenals, thymus and ovaries) were statistically significant, when compared with the concurrent Control. Differences from Control considered most likely to be of toxicological significance were restricted to increased liver weight, which was also apparent for females at 5000 ppm and decreased adrenal, thymus and, to a lesser extent, spleen weight. Excluding the increased liver weight observed at 5000 ppm, organ weights of females at this dosage and at 1000 ppm were not obviously affected by treatment.

Histopathology of adults
Histopathological findings in the F1 generation considered to be related to treatment were restricted to changes in the livers of males at 5000 ppm and of both sexes at 10000 ppm. The range of findings observed for the high dosage group males is indicative of hepatotoxicity of the periacinar hepatocytes with consequential increased cell turnover resulting in regenerative hyperplasia. The development of altered cell foci in males at this dosage is probably a consequence of the increase turnover of hepatocytes, while bile duct proliferation is probably associated with the altered architecture of the liver. Changes in the liver may be associated with an increase in peroxisomal enzymes, which has been demonstrated in the livers of five treated F1 animals at this dosage. Males at 5000 ppm and females at 10000 ppm livers were less affected and showed periacinar hepatocytic vacuolation, periacinar hepatocytic hypertrophy and periacinar hepatocytic necrosis; the females in particular showing only a low incidence of findings. There were no other microscopic findings observed that were considered to be related to treatment.

Palmitoyl CoA Oxidase activity in male livers
Assessment of the livers from five F1 male from each group at termination showed a statistically significant, and treatment related, increase in palmitoyl CoA oxidase activity in male rats which received the substance. This provides some evidence that the substance was acting as a relatively weak peroxisome proliferator under the conditions of the study.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 10 000 ppm
Based on:
test mat.
Sex:
male/female

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
Description (incidence and severity):
decease
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
male liver
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
increased liver cell turnover

Details on results (F1)

F0 generation
Offspring bodyweight
Bodyweights of male and female offspring at Day 1 of age was unaffected by treatment. At 10000 ppm overall bodyweight gain of both sexes to weaning at Day 21 of age was marginally lower than Controls, although these differences failed to attain statistical significance, principally due to a slightly lower mean gain between Day 7 and 14 of age. A similar effect on bodyweight gain during this period was evident in the F2 offspring. At 1000 and 5000 ppm bodyweight gain of both sexes to weaning was essentially similar to Control.

Necropsy of offspring
Necropsy of offspring which died before weaning revealed absence of milk in the stomach as the only consistent finding. This finding is commonly seen in neonates which die at an early age. Neither the type, incidence or distribution of findings at necropsy of offspring at 25 days of age indicated any adverse effect of treatment with the substance.

Organ weights of offspring
At 10000 ppm, although mean bodyweight was slightly below that of the Control counterparts, there was a marked increase in mean absolute liver weight for both sexes when compared to Control, differences attaining statistical significance. This difference was very evident when the organ weight was expressed as a percentage of the offspring bodyweight. A similar increase in mean liver weight of the offspring was also observed for the F2 generation. At 5000 ppm absolute liver weight for both sexes was slightly higher than the concurrent Control; further divergence from Control was again evident when organ weight was expressed as a percentage of bodyweight. Liver weight at 1000 ppm was similar to the Control.
There was no obvious adverse effect of treatment on the absolute or relative organ weight of the brain, spleen or thymus for either sex at any of the dosages investigated. Lower thymus weight was noted at 5000 and 10000 ppm for both sexes, however, as organ weight expressed as a percentage of bodyweight values were essentially similar to control, there was no conclusive association with treatment.

F1 generation
Offspring bodyweight
Bodyweights of male and female offspring at Day 1 of age was unaffected by treatment at any dosage investigated.
At 10000 ppm overall bodyweight gain of both sexes to weaning at Day 21 of age was lower than Control, these differences attaining statistical significance by Day 14, and as in the F0 generation the greatest divergence from the control appeared to occur between Day 7 and 14 of age. At 5000 ppm overall bodyweight gain of both sexes to weaning at Day 21 of age was lower than Control, differences attaining statistical significance by Day 14. This was attributable to lower gain until Day 14 of age, subsequent gain was similar to Control. At 1000 ppm bodyweight gain of both sexes to weaning was generally similar to Control.

Necropsy of offspring
Necropsy of offspring that died before weaning revealed absence of milk in the stomach as the only consistent finding. This finding is commonly seen in neonates who die at an early age. Neither the type, incidence or distribution of findings at necropsy of offspring at 25 days of age indicated any adverse effect of treatment.

Organ weights of offspring
Assessment of organ weights at 10000 ppm amongst F2 offspring was complicated by the significant reduction in bodyweight in both sexes at this dosage. There was a decrease in the weight of the spleen and thymus and an increase in the weight of the liver in both sexes, these differences attaining statistical significance. However, when expressed as a percentage of bodyweight, the inter-group differences disappear for spleen and thymus and therefore the toxicological significance of these findings may be of some doubt. The relative organ weight of the liver however, diverges further from the control and it is likely that the effect observed on absolute weights for this organ in both sexes is of toxicological importance. The absolute organ weight of the brain at 10000 ppm was similar to the Control, and, as absolute weight is considered to be a more accurate indicator of effect on the brain than relative weight, no effect of treatment is indicated. At 5000 ppm there was an increase in both the absolute and relative weight of the liver, differences in absolute weight attaining statistical significance. Neither the brain, spleen or thymus were considered to have been affected by treatment at this inclusion level. There was no obvious adverse effect on either the absolute or relative weights of the brain, liver, spleen or thymus at 1000 ppm.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 10 000 ppm
Based on:
test mat.
Sex:
male/female

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 10 000 ppm
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It was concluded that the substance had no adverse effects upon fertility or reproductive performance when rats were exposed to diets containing up to 10000 ppm of the test material through two successive generations.
Executive summary:

The influence of the substance on the fertility and reproductive capacity of two successive generations was assessed in male and female rats. The substance was administered continuously in the diet at concentrations of 1000, 5000 or 10000 ppm to groups of rats throughout the two generations. The highest dosage group animals were given 20000 ppm of D911P for approximately six weeks (43 days) at the start of the Fo generation but this was reduced to 10000 ppm because of a marked reduction in bodyweight gain of the males. The substance had no adverse effects upon fertility or reproductive performance when rats were exposed to diets containing up to 10000 ppm of the test material through two successive generations. The substance had no adverse effect on the seminology parameters investigated and no histopathological effects on the reproductive organs.10000 ppm in the diet was considered to be the no-adverse-effect-level for fertility and reproductive performance in the rat.