1,2-Benzenedicarboxylic acid, di-C9-11-branched and linear alkyl esters

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 24,1997 to June 7, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to recognised test methods

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited
- Age at study initiation: 6 weeks
- Weight at study initiation: 145 - 228 g (males) , 115-198 g (female)
- Fasting period before study: Not applicable
- Housing: Group caged (4 animals of the same sex) except during mating (1 male/1 female per cage) and, for females, during gestation, littering and weaning (1 female + litter per cage).
- 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): 19 - 21°C
- Humidity (%): 42-71%
- Air changes: Approximately 15/hour
- Photoperiod : 12 hours dark/light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet: Weekly
- Mixing appropriate amounts with (Type of food): A pre-mix of diet was prepared by weighing out the quantity of test substance 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 for a minimum period of 6 minutes (for at least 100 cycles). The required dietary concentrations were prepared by direct dilution of the premix with further quantities of untreated diet. Blending was achieved by mixing for a minimum period of 6 minutes (for a least 100 cycles).
- Storage temperature of food: Ambient conditions

VEHICLE
- Justification for use and choice of vehicle: Dietary administration was selected to simulate the potential conditions of human exposure
- Concentration in vehicle: 0 ppm (control), 1000 ppm, 5000 ppm and 10000 ppm. 20000 ppm was originally selected as the initial concentration for the highest dosage group, 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: Up to 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged: Individually
- Any deviations from standard protocol: No

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 analytical procedure validation, the homogeneity and stability during ambient temperatute storage for 22 days was confirmed for the test substance in the diet at nominal concentrations of 100 ppm and 20000 ppm.

Single samples (nominally 200g) were taken from all groups/sex (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.

Duration of treatment / exposure:

For this purpose D911P 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 the substance in the diet for approximately six weeks (43 days) at the start of the F0 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 F0 generation received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation. From the F1 litters offspring were selected to form the F1 generation, both sexes of which 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 which was available ad libitum.

Details on study schedule:

- F1 parental animals not mated until 10 weeeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21-28 days of age.
- Age at mating of the mated animals in the study: 14 weeks

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

No. of animals per sex per dose:

28 males and 28 females/dose group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on outcome of a preliminary study
- Rationale for animal assignment: Randomised following body weight stratification, ensuring 1 male/1 female from each litter was present in each group

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations included: Deviations from normal.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Males - weekly. Females - weekly until mating, gestation days 0, 6, 13 and 20, lactation days 1, 4, 7, 14 and 21.

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):

Vaginal smears taken for 10 days before pairing to establish teh duration and regularity of the oestrus cycle.

Sperm parameters (parental animals):

Parameters examined in all (P and F1) male parental generations: testis weight, epididymis weight, sperm motility, sperm count in epididymides, sperm morphology, homogenisation resistant spermatids, microscopic pathology of epididymides and testis

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 and F2 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/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals killed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals killed after the last litter of each generation was weaned.

GROSS NECROPSY
- Detailed gross necropsy consisted of external and internal examinations including the cranial, thoracic, abdominal and pelvic cavities and their viscera.

ORGAN WEIGHTS
The following tissues from parental animals were weighed: adrenal glands, brain, epididymides, kidneys, liver, ovaries, prostate, seminal vesicles and goagulating gland, spleen, testes, thymus, uterus with cervix

HISTOPATHOLOGY
The following tissues/organs from parental animals were preserved for histopathology: abnormalities, adrenal glands, brain, epididymides, kidneys, liver, mammary glands, ovaries, oviduct, pituitary, prostate, seminal vesicles and goagulating gland, spleen, testis (right), thymus, uterus with cervix, vagina

Postmortem examinations (offspring):

SACRIFICE
- F1 animals not selected for continuation of the study were killed at 25 days of age. F2 offspring were killed after weaning at 25 days of age.

GROSS NECROPSY
- Gross necropsy consisted of examination for evidence of disease or adverse reaction to treatment.

ORGAN WEIGHTS
The following tissues from unselected F1 and F2 animals were weighed: brain, liver, spleen, thymus

HISTOPATHOLOGY
The following tissues from unselected F1 and F2 animals were preserved for histopathology: abnormalities, brain, epididymides, liver, ovaries, oviduct, prostate, seminal vesicles and goagulating gland, spleen, testes, thymus, uterus with cervix, vagina

Statistics:

Significance tests , employing of variance followed by an inter-group comparison with the control, were performed on the following parameters: Bodyweights and bodywheight range.

Reproductive indices:

Oestrus cycle, pre-coital interval, mating performance and fertility, gestation length, litter size, sex ratio, sperm analyses

Offspring viability indices:

Post-implanation survival, liver birth index, viability index, lactation index

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

bodyweight decrease at 20000/10000 ppm

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

bodyweight decrease at 20000/10000 ppm

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver at 20000/10000 ppm

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: reduced in males at 20000/10000 ppm

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
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 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). Necropsy failed to reveal an obvious cause for the animal's condition. One female receiving 10000/20000 ppm was killed during Week 16 after showing apparent bleeding from the vagina 2 days after parturition. Necropsy findings were unremarkable. The incidence and circumstances of the deaths of the two treated females did not indicate any association with treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
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. Mean bodyweight gain of males at 5000 and 1000 ppm were not adversely affected by treatrnent and were generally comparable to those of 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; however, 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. The significance of this finding is, again, 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.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
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.
Overall the intended 5 and 4/2 fold differences between treatment groups was maintained at any given point throughout the study but, with the lowering of the interval between the intermediate and high dosage group, there were times when animals within the intermediate group reached intake levels
approaching those of the highest dosage at a different stage of the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
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.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
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 these was no evidence of an effect of treatment upon the homogenisation resistant spermatids within the testis. It was noted that single males from the Control), low- and mid-dose groups were siblings and all three showed a low percentage of progressively motile sperm and failed to induce pregnancy in their female oartners.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was no adverse effect of treatment on pre-coital interval, the majority of animals mating within four days of pairing i.e. at first oestrus opportunity. Fertility was not affected by treatment at any of the dosages investigated.

ORGAN WEIGHTS (PARENTAL ANIMALS):
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.

GROSS PATHOLOGY (PARENTAL ANIMALS):
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 had 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.

HISTOPATHOLOGY (PARENTAL ANIMALS):
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 dosage group males is indicative of hepatoxicity 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.

OTHER FINDINGS (PARENTAL ANIMALS)
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.

Dose descriptor:

NOAEL

Effect level:

ca. 10 000 ppm

Based on:

test mat.

Sex:

male/female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

deceased gain to weaning at 10000 ppm

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver at 10000 ppm

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

male liver

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

liver, males at 5000 and 10000 ppm

VIABILITY (OFFSPRING):
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 Groups 1 to 4 respectively), 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.

CLINICAL SIGNS (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.

BODY WEIGHT (OFFSPRING):
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 marginally lower than Control (although 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.

SEXUAL MATURATION (OFFSPRING):
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.
There was no obvious adverse effect on the regularity and duration of the oestrus cycles and no adverse effect of treatment on pre-coital interval on mating.


ORGAN WEIGHTS (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 and further divergence from Control was again evident when organ weight was expressedes a pereontage 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 thymns for either sex at any of the dosages investigated Loweethyinus weight was hosed at 5000 and 10000 pprn for both sexes however, as organ weight expressed as á percentage of
bodyweight values were essentially similar to control; there was no conclusive association with treatment.

GROSS PATHOLOGY (OFFSPRING):
Necropsy of offspring that died before weaning revealed the absence of milk in the stomach as the only consistent finding, this being commonly seen in neonates who die at an early stage. Neither the type, incidence or distribution of findings at necropsy of offspring at 25 days of age indicated any adverse effects of treatment.

HISTOPATHOLOGY (OFFSPRING) 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 particuler showing only a low incidence of findings. There were no other microsscopical findings observed that were considered to be related to treatment..

OTHER FINDINGS (OFFSPRING)
Sex ratio at birth (as assessed by percentage males) was not influenced by treatment and there was no selective effect on pup survival of either sex.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 10 000 ppm

Based on:

test mat.

Sex:

male/female

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

ca. 10 000 ppm

Based on:

test mat.

Sex:

male/female

Reproductive effects observed:

not specified

Conclusions:

It was concluded that the tested 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.

Executive summary:

The influence of Diplast L9 -11 on the fertility and reproductive capacity of two successive generations was assessed in male and female rats using methods prescribed by OECD test guidelines. 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 for approximately six weeks (43 days) at the start of the F0 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

650 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation fertility study has been undertaken. In this study no adverse effects upon fertility or reproductive performance when rats were exposed to diets containing up to 10000 ppm, corresponding to a mean daily intake of at least 650 mg/kg/day, through two successive generations. D911P fed through two generations had no adverse effect on the seminology parameters investigated and no histopathological effects on the reproductive organs.

Short description of key information:
Fertility: NOAEL - 650 mg/kg/day

Effects on developmental toxicity

Description of key information

Developmental toxicity: NOAEL - 1000 mg/kg/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from September 29,1999 to October 28,1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted to recognised international test guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River (UK) Ltd, Margate, Kent
- Justification: the rat was chosen because of its use as a predictor of reproductive toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in this laboratory.
- Age at study initiation:10 - 11 weeks
- Weight at study initiation:220-267 g
- Housing:barriered limit access, rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical ag ents and to minimise the transference of such agents between rooms. Access was limited to authorised personnel who were required to shower and change into clean protective clothing. Where practicable, materials and equipment entered the facility through an autoclave or a chamber in which th eir external surfaces were treated with a bactericide. Before the study commenced the room was cleaned and disinfected with a bactericide.
- Diet :ad libitum
- Any other information about food:commercially available pelleted expanded rodent diet (DSD laboratory animal diet N°1) from special diet services Limited, periodical analysed by supplier.
- Water :ad libitum:
- Acclimation period:5 days
- Medical check: during the acclimatattion period , twice a day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/- 4 °C
- Humidity (%): 55% (48% - 61%)
- Air changes (per hr):15 room air changed per hour
- Photoperiod :12 hour cycle dark/lightm on 6:00 GMT

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prapared on a weekly basis by hand mixing the test substance with the selected vehicle followed by homogenisation. The prepared formulations were divided into daily aliquots which were stored at ambient temperature

VEHICLE
- Justification for use and choice of vehicle:The oral (gavage) route was selected to simulate the conditions of possible human exposure
- Concentration in vehicle: 0, 50, 100, 200 mg/ml (to give dose levels of 0, 250, 500, 1000 mg/kg/day)
- Amount of vehicle (if gavage): 5 ml/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration on one occasion during the first and last weeks of treatment were analysed for test material content. Four 1.0 ml sample were taken from each group on each occasion 2 samples per group were analysed for test material content and the
remaining 2 were retained as contingency samples.

Analysis showed concentrations to be within +3.8% / -1.8% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: 3 x vaginal plugs or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

19 days (Days 1-19 of gestation)

Frequency of treatment:

Daily

Duration of test:

20 days (gestation)

Remarks:

Doses / Concentrations:
0
Basis:
nominal conc.
= dose level of 0mg substance / kg b.w. (control)

Remarks:

Doses / Concentrations:
50 mg/ml
Basis:
nominal conc.
= dose level of 250mg substance / kg b.w.

Remarks:

Doses / Concentrations:
100 mg/ml
Basis:
nominal conc.
= dose level of 500mg substance / kg b.w.

Remarks:

Doses / Concentrations:
200 mg/ml
Basis:
nominal conc.
= dose level of 1000mg substance / kg b.w.

No. of animals per sex per dose:

Three groups of 22 females + a control group (22 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on outcome of a preliminary study
- Rationale for animal assignment (if not random): Allocated to group/cage position in sequence of mating. Allocation adjusted to prevent to prevent any stock male providing more than one mated female in each treatment group. Allocation adjusted if Day 0 group mean body weights differed beyond acceptable limits.
- Other: Cage distribution arranged to minimise possible environmental variables

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes , Females were weighed and bodyweight recorded daily throughout the study. Bodyweights Days 0, 1, 3, 6, 9, 12, 15, 18 and 20 after mating were rported.

FOOD CONSUMPTION : Yes , food consumption recordings were made for the periods Days 1-2, 3-5, 6-8, 9-11, 12-14, 15-17 and 18-19 after mating.

POST-MORTEM EXAMINATIONS: Yes

- Organs examined: yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of distribution of foetuses in each uterine horn: yes

Fetal examinations:

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

Foetuses were identified within the litter with respect to their uterine position. Each foetus was weighed, sexed and examined for any external abnormalities. individual placental weights and placental abnormalities were recorded. Foetuses were then killed. Approximately half of each litter was allocated to fresh visceral examination, were eviscerated and fixed before processing staining with Alizarin Red, using a modification of the Dawson staining technique, for examination of their skeletons. The remaining foetuses were fixed in Bouin's fluid before examination by the Wilson free-hand serial sectioning technique.

Statistics:

Where appropriate, data was subjected to statistical analyses. Dependent on the heterogeneity of variance between groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams' 1971/2) or non-parametric tests, (Kruskal-Wallis, Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate. For litter data the basic sample unit was generally the litter and, due to preponderance of non-normal distributions, non-parametric analyses were routinely used.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

MATERNAL RESPONSAL:

Clinical signs

Increased levels of salivation after dosing were seen sporadically in all groups, including control. A greater number of animals were affected at 1000 mg/kg/day than in the Control and lower dosage groups; however, it was noted that post-dosing salivation is frequently observed in studies with oral gavage dosing and its occurrence is generally considered to reflect distaste of the dosing formulation rather than

indicating toxicological effect. Several animals in all groups (including control) showed staining of the coat on the head and body, and some incidences of hairloss: these observations were considered to be related to the use of oil as the dosing vehicle and to be of no toxicological significance.

Bodyweight

Bodyweights and bodyweight gain during gestation were unaffected by treatment. Adjustment of overall bodyweight gain for the contribution of the gravid uterus showed no adverse effect of treatment on bodyweight gain.

Food consumption

Food consumption during gestation was unaffected by treatment; group mean values for all treated groups were similar to the Control throughout

Macroscopic pathology

Macroscopic findings at neuropsy of females on Day 20 of gestation were unremarkable being generally restricted to confirmation of signs (hairloss staining) observed in-life. No findings considered to be of toxicological significance were observed.

LITTER RESPONSES:

Litter data

All females were pregnante , however one female at 1000 mg/kg/day was found to only have three empty implantation sites (two in the left and one in the right uterine horn). This litter was classified as total litter resorption (total litter loss in-utero). Given the isolated nature of this finding, and the absence of any adverse effect on the process of implantation (as judged by pre-implantation loss) or on foetal survival (as judged by post-implantation loss), an association with treatment was considered unlikely.

The number of implantations, resorptions and live foetuses were essentially similar in all groups and were unaffected by treatment. There was no evidence that the implantation process or in-utero survival had been adversely affected by administration of the test substance. Sex ratio, expressed as percentage males, was comparable in all groups.

Placental, litter and foetal weights

There were no obvious adverse effects of treatment on placental, litter or foetal weights.

It was noted that mean foetal weight was higher in both sexes at 1000 mg/kg/day, with differences from Control attaining statistical significance for females; this was not considered to be of any toxicological significance.

Foetal evaluation

There were 3, 1, 2 and 3 foetuses (3, 1, 2 and 2 litters affected) in Groups 1 to 4 respectively showing major abnormalities. Neither the type, incidence nor distribution of major abnormalities indicated any obvious adverse effect of treatment with the tested substance.

The incidence and distribution of minor visceral and skeletal abnormalities or skeletal variants did not indicate any adverse effect of treatment on foetal development.

At 1000 and 250 mg/kg/day, detailed visceral examination revealed a higher incidence of foetuses showing dilated ureter compared to the Control, although this incidence was within the recent background control data range. Accompanying this finding was a higher incidence of renal cavitation and hydroureter in animals allocated to skeletal processing, at these dosages compared to the Control.

Collectively these findings were noted to cluster, with two litters at 250 mg/kg/day and two litters at 1000 mg/kg/day being particularly affected. In view of the litter clustering and the absence of any clear dosage relationship, these findings were considered to be coincidental, and emphasised by a low incidence in the Control group.

At 1000 and 500 mg/kg/day there was also an increased incidence of foetuses with 13/14 or 14/14 ribs. Again there was no dose relationship and, as the total number of litters affected remained similar to that of the Control group, it was considered that this finding could not, with certainty, be attributed to treatment.

Conclusions:

Potential developmental toxicity has been studied in the rat using methods in accordance with OECD TG414. Daily treatment from before implantation to just before the expected day of parturition resulted in no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development at dose levels up to 1000 mg/kg bw/day.

Executive summary:

Potential developmental toxicity has been studied in the rat using methods in accordance with OECD TG414. Daily treatment from before implantation to just before the expected day of parturition resulted in no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development at dose levels up to 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A study of developmental toxicity has been undertaken. In this study no clear adverse effects on pregnancy of the female rat or on embryofoetal survival and development were apparent at dose levels up to 1000 mg/kg bw/day.

Justification for classification or non-classification

Classification is not justified based on the available information from a 2 -generation reproductive toxicity study and a study of developmental toxicity. This justification is supported by the ECB decision on the substance regarding classification and labelling (Meeting of the Commission Working Group on the Classification and Labelling of Dangerous Substances, ECB Ispra 16 -18th January 2002, ECBI/15/02 Rev.3).