[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 December 2008 to 17 April 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP compliant; no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: (P) 12 wks; (F1) x wks
- Weight at study initiation: (P) Males: 303-361 g; Females: 183-224 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2
- Humidity (%): 55±15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: A known amount of test material was mixed with a small amount of basal laboratory diet for nineteen minutes at a constant speed, setting 1 in a Hobart QE200 mixer. This premix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.


DIET PREPARATION
- Rate of preparation of diet: Prior to treatment and once during the study period
- Mixing appropriate amounts with: Rodent PMI 5002 (Certified) Ground Diet (BCM IPS Limited, London, UK)
- Storage temperature of food: The diet was stored at ambient temperature in labelled, double blue plastic bags in labelled, covered plastic bins when not in use.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: the presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)
- After successful mating each pregnant female was caged individually in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes
- Any deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dietary admixtures were extracted with methanol to give a final, theoretical test material concentration of approximately 25 ppm. Standard solutions of test material were prepared in methanol at a nominal concentration of 25 ppm.

The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC Agilent Technologies 1200, incorporating autosampler and workstation
Column Microsorb-MV 100-A 5 µm C18 (150 x 4.6 mm id)
Mobile phase Acetonitrile:0.1% orthophosphoric acid (85:15 v/v)
Flow-rate 1 ml/min
UV detector wavelength 215 nm
Injection volume 10 µl
Retention time ~3.5 mins

The dietary admixtures were sampled from the middle and two opposite sides in triplicate and analysed. The dietary admixtures were sampled and analysed initially and then after storage at ambient temperature in the dark for six weeks. The dietary admixtures were sampled and analysed within four days of preparation.

Duration of treatment / exposure:

45 days

Frequency of treatment:

Daily

Details on study schedule:

No further details

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 5000 and 20000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Based on previous toxicity work
- Rationale for animal assignment: Not applicable

Positive control:

None

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily


BODY WEIGHT: Yes
Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Day 0, 7, 14 and 20 post coitum, and on Day 1 and 4 post partum.


FOOD CONSUMPTION AND COMPOUND INTAKE:
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1 - 4).
Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase and during the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

Oestrous cyclicity (parental animals):

Yes, every morning

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No (screening)


PARAMETERS EXAMINED
The following parameters were examined in offspring: Group mean corpora lutea and implantation counts, litter size, implantation, survival indices and sex ratio


GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The male dose groups were killed on Day 43.
- Maternal animals: At Day 5 post partum, all surviving females were killed.


GROSS NECROPSY
All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.


HISTOPATHOLOGY / ORGAN WEIGHTS
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin: Coagulating gland, epididymides, ovaries, mammary tissue (females only), pituitary, prostate, seminal vesicles, testes, uterus/cervix, and vagina.

Postmortem examinations (offspring):

SACRIFICE
At Day 5 post partum, all surviving offspring were killed.


GROSS NECROPSY
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

No information

Reproductive indices:

Yes

Offspring viability indices:

Yes

Results and discussion

Results: P0 (first parental generation)

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

20 000 ppm

Sex:

male/female

Basis for effect level:

other: no effects

2

Dose descriptor:

NOAEL

Effect level:

1 952 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: calculation based on data from report

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Slightly lower numbers of corpora lutea counts and implantation sites were evident for females treated with 20000 ppm when compared to controls and slightly higher postimplantation losses were also evident at the highest dose in comparison to controls. This resulted in slightly smaller litter sizes at birth at 20000 ppm in comparison to litter sizes at birth from controls (group mean value of 9.6 for 20000 ppm compared to a group mean value of 11.3 for controls). Statistical analysis of the data however did not reveal any significant intergroup differences.
There were no differences in live birth indices between control and treated groups, although offspring viability was lower for 20000 ppm when compared to controls, which was attributable to the loss of a majority of offspring from one 20000 ppm litter. Furthermore, there were higher percentages of males born per litter for treated animals when compared to controls, although statistical significance was not achieved.


CLINICAL SIGNS (OFFSPRING)
Daily clinical observations of litters revealed interim deaths of offspring from two litters at 20000 ppm. Four offspring were found dead from one 20000 ppm litter on Pre-Day 1 and the majority of offspring from another 20000 ppm litter was missing (cannibalised by the mother) on Day 2 post parium. There were no interim deaths of offspring observed in the remaining treatment groups or in the controls.


BODY WEIGHT (OFFSPRING)
Lower total litter weights were also detected at 20000 ppm when compared to controls. Furthermore, female offspring from the 20000 ppm litters showed a 17% less bodyweight gain during lactation when compared to controls during the lactation period. Statistical analysis of the data, however, did not result in any significant intergroup differences and individual offspring weights were not affected. No adverse effects on bodyweight change were detected for male offspring from litters from treated groups when compared to controls.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 000 ppm

Sex:

male/female

Basis for effect level:

other: significant effects on viability, clinical signs and body weight

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

484 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: calculation based on data from report

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The results of this study were reviewed by the Head of Toxicology of Harlan Laboratories, and the following comments were made (full report attached as background material in "0656-0395 Evaluation of Findings (finalised).pdf":

 

This further evaluation puts into context the key findings seen in the Oral (Dietary)

Reproduction/Developmental Toxicity Screening test in the rat for Experimental Additive

20735-82 (Harlan Laboratories Project Number 0656/0395). In summary there were a number of highlighted differences between reproduction values for high dose animals against controls. The background control data for this strain of animal is attached. It should be of note that the high dose level of 20000 ppm equated to a test material intake of above 1000 mg/kg particularly during lactation for females. The test material showed no significant evidence of toxicity to adult animals but the volume of chemical intake may well have impacted upon the nutritional intake from the diet consumed thereby providing less than optimal conditions for females during pregnancy and early lactation.

The actual findings of note are summarised as follows:

 

1. Mating performance/pregnancy.

At 20000 ppm there were two females that failed to produce offspring. The two events are of different origin in that one male/female pairing showed no positive evidence of mating during a 14 day cohabitation period. The other male/female pairing showed positive evidence of mating but failed to produce a litter of offspring.

Both of these events are observed to occur spontaneously at a low level on reproduction studies. Failure to show evidence of mating can be a result of female pseudopregnancy when the process of vaginal lavage can actually induce this process which then results in the female remaining acyclic for up to 16 days. A failure of conception can also occur spontaneously and quite often there is no indication of aetiology. This is also potentially the case for the male/female pairing at 5000 ppm which failed to produce a pregnancy, although in this situation the testicular atrophy for male 44 may well be a contributory factor. No such significant histopathological changes were seen at 20000 ppm and so the failure of conception remains unclear as to whether this is truly a treatment-related effect or a spontaneous event.

As a result of the two females without offspring a lower group number of pregnant females were available for analysis of litter/reproduction parameters.

 

2. Mean corpora lutea and implantation sites

The mean value for corpora lutea counts for females at 20000 ppm was lower than the equivalent values for concurrent controls. Two females showed corpora lutea values that were outside the present background control range. Both values were however not outside what can occasionally happen where a female will produce a lower than expected litter size. The number of implantations are entirely a consequence of the lower corpora lutea count for these females and not a result of a pre-implantation embryo loss. The corpora lutea counts for the remaining six females were unremarkable which does question whether the effect seen was truly significant if larger group sizes were to be evaluated.

 

3. Post-implantation loss

A slightly higher mean value for post implantation embryo loss was reported for high dose group females compared with control values albeit the difference is within one standard deviation of control values. There was no obvious dose-related trend and the value achieved was at the high end of the range quoted for background control values but above the mean value. An evaluation of individual female values for the high dose group shows no obvious abberant values and so this seems a small variation without any obvious reason. Individual values for post implantation embryo loss do not demonstrate a toxicologically significant variation from what would commonly be expected to be seen in this type of study.

 

4.Post partum female offspring bodyweight gain

At 20000 ppm there was a slight reduction in mean bodyweight gain for female offspring compared with control values. This reduction in weight gain resulted in a 0.4 gram difference in group mean values compared with control values which was within 1 standard deviation of control mean values and was also within background control range. No effect was seen with male offspring at this dose level compared with control values. This difference may well represent a chance result. In addition, there was no difference in offspring bodyweight values at birth compared with controls. This reported effect may therefore be a chance result.

 

Conclusions

The administration of this test material at dose levels above 1000 mg/kg intake by dietary inclusion showed the material to be of low toxicity to the adult. Because test material intake increased significantly during pregnancy and lactation this may have contributed to the minor differences in reproduction parameters measured. The failure to mate or conceive was seen in isolated individuals. These events do occasionally occur as a spontaneous event and because it was seen in one individual for each of the categories it cannot be considered a convincing effect of treatment. Minor changes in corpora lutea counts, implantation numbers and post implantation loss values are not conclusive evidence of a specific reproductive effect when considered in isolation. Collectively, these findings together with the slight reduction in female offspring weight gain during the first four days of lactation may well reflect minor fluctuations in reproductive performance as a consequence of elevated body burden of test material above 1000 mg/kg day. No one clear reproductive event seems to have been affected to suggest the test material is a reproductive toxicant and therefore this screening study has not shown the test material to significantly affect reproductive and developmental behaviour.

Applicant's summary and conclusion

Conclusions:

The administration of this test material at dose levels above 1000 mg/kg intake by dietary inclusion showed the material to be of low toxicity to the adult. Because test material intake increased significantly during pregnancy and lactation this may have contributed to the minor differences in reproduction parameters measured. The failure to mate or conceive was seen in isolated individuals. These events do occasionally occur as a spontaneous event and because it was seen in one individual for each of the categories it cannot be considered a convincing effect of treatment. Minor changes in corpora lutea counts, implantation numbers and post implantation loss values are not conclusive evidence of a specific reproductive effect when considered in isolation. Collectively, these findings together with the slight reduction in female offspring weight gain during the first four days of lactation may well reflect minor fluctuations in reproductive performance as a consequence of elevated body burden of test material above 1000 mg/kg day. No one clear reproductive event seems to have been affected to suggest the test material is a reproductive toxicant and therefore this screening study has not shown the test material to significantly affect reproductive and developmental behaviour.

Executive summary:

In an Oral (dietary) reproduction/developmental toxicity screening test in the rat (OECD 421, 1995) Experimental Additive 20735-82 was administered to three groups each of ten male and ten female Wistar Han:HsdRccHan:WIST strain rats in dietary admixture at dose levels of 20000, 5000 and 250 ppm (equivalent to a mean dosage of 1165, 296 and 14 mg/kg/day for males and 1533, 411 and 20 mg/kg/day for females). 

 

In summary there were a number of highlighted differences between reproduction values for high dose animals against controls. The background control data for this strain of animal is attached. It should be of note that the high dose level of 20000 ppm equated to a test material intake of above 1000 mg/kg particularly during lactation for females. The test material showed no significant evidence of toxicity to adult animals but the volume of chemical intake may well have impacted upon the nutritional intake from the diet consumed thereby providing less than optimal conditions for females during pregnancy and early lactation.

 

The administration of this test material at dose levels above 1000 mg/kg intake by dietary inclusion showed the material to be of low toxicity to the adult. Because test material intake increased significantly during pregnancy and lactation this may have contributed to the minor differences in reproduction parameters measured. The failure to mate or conceive was seen in isolated individuals. These events do occasionally occur as a spontaneous event and because it was seen in one individual for each of the categories it cannot be considered a convincing effect of treatment. Minor changes in corpora lutea counts, implantation numbers and post implantation loss values are not conclusive evidence of a specific reproductive effect when considered in isolation. Collectively, these findings together with the slight reduction in female offspring weight gain during the first four days of lactation may well reflect minor fluctuations in reproductive performance as a consequence of elevated body burden of test material above 1000 mg/kg day. No one clear reproductive event seems to have been affected to suggest the test material is a reproductive toxicant and therefore this screening study has not shown the test material to significantly affect reproductive and developmental behaviour.