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Diss Factsheets

Administrative data

Endpoint:
one-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:
09 October 1995 to February 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU & OECD test guidance in compliance with GLP. The study is read across to an analogous substance; refer to image and further information below.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.34 (One-Generation Reproduction Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
H112323
IUPAC Name:
H112323
Details on test material:
See below

Test animals

Species:
rat
Strain:
other: Alpk'APfSD
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species: Rat
Strain: AIpk:APfSD
Source: Barriered Animal Breeding Unit, Zeneca Pharmaceuticals, Alderley Park
Sex: 28 litters of 4 males per litter and 28 litters of 4 females per litter
Specification: Specific Pathogen Free (SPF). Supplied as weanlings (approximately 22 days old) segregated by sex and litter of origin
Accommodation and husbandry: The rats were housed, sexes separately, in multiple rat racks suitable for animals of this strain and weight range expected during the course of the study. They were housed in litters initially, and two males or two females per cage after they had been assigned to experimental groups and during the pre-mating period- females were housed individually during pregnancy and lactation and provided with bedding material.

The rats were transferred to clean cages and racks, as necessary, during the study

The animal room was designed to give the following environmental conditions.
Temperature: 21±2°C
Relative humidity: 55+15%
Air: At least 15 changes/hour, giving positive pressure with respect to the access corridor
Light cycle: Artificial giving 12 hours light, 12 hours dark

Diet (CTI) and mains water generally supplied by an automatic system were available ad libitum. Water was supplied by water bottles during late pregnancy and gestation.

Each batch of diet is routinely analysed for composition and for the presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.

Acclimatisation
The animals were housed under experimental (SPF) conditions for at least 5 days at CTL, prior to the start of the study For 10 days following delivery of the rats, personnel access to the animal room was restricted as a quarantine procedure.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Not applicable.
Details on exposure:
The experimental diets were prepared in or 60kg batches by direct addition of HI 12323 to CT1 diet and were mixed thoroughly.

Diets were prepared by means of direct addition of weighed amounts of the test substance to CT1 diet and mixing for 6 minutes in a Pharma Matrix blender. The amounts of test substance were as shown below:

Group Dietary concentration of HI 12323 (ppm) Amount of HI 12323 for 60kg Diet Batch (g)
1 0 (control) 0
2 1000 60
3 5000 300
4 20000 1200

Diets were dispensed into jars and stored at room temperature. Fresh jars of diet were given to the animals as required.
Details on mating procedure:
Mating
Females were mated with males of the same group, but in all pairings, brother-sister mating was avoided.
During mating, one male was replaced with a female from the adjacent cage thus, one male was housed with one female Vaginal smears were examined daily to determine when mating occurred (as shown by the presence of sperm)

Pregnancy and lactation
Any female with a positive smear was immediately separated from the male and individually housed.

Reproductive performance
From the records of mating and parturition, the reproductive performance of the adults was assessed. The following were examined:

The proportion of successful matings. The criterion for a successful mating was the production of a viable litter i. e. a litter in which at least one pup was found alive at day 1.

Length of gestation was measured in days from the date of the positive smear to date of birth (but only in females fulfilling the criterion above i e production of a viable litter)

Pre-coital interval, in days between the date of pairing and the date of the positive smear, was measured.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SUMMARY - SPECTROPHOTOMETRY
Samples were extracted with methanol Portions of supernatant were diluted with methanol, as appropriate, to give sample solution concentrations within the range of calibration standards and analysed by Ultra Violet/Visible Spectrophotometry (UV/VIS)

CHEMICALS AND REAGENTS
Methanol, Analytical grade

CALIBRATION STANDARDS

Analytical standard
HI 12323, CTL Reference Y0696S/004, with an assumed purity of 100% was used (test substance).
Nominally 100mg of test substance was accurately weighed into a volumetric flask and diluted to volume with methanol (nominally 1.0mg/ml)
Further appropriate dilutions were made with methanol in volumetric flasks to give an appropriate range of solutions, nominally within 5μg/ml to 100μg/ml


PROCEDURE

Sample preparation
Accurately weighed portions of diet samples, 5g, were added to tared conical flasks, 50ml of methanol added, and the flasks stoppered. Samples were extracted using mechanical

RECOVERY
For each analysis, recovery was determined in triplicate at each level.

20000ppm level
An accurately weighed portion of test substance, 1g, was quantitatively transferred into a mortar with 49g of control diet and ground for 5 minutes. The diet was placed in a suitable container and rotated on the Stuart flask rotator for 30 minutes at an appropriate speed

5000 and 1000ppm levels
Accurately weighed portions of test substance, 250 and 50mg, were quantitatively transferred into a mortar with 49 75 and 49,95g respectively control diet, and ground for 5 minutes The diets were placed in a suitable container and rotated on the Stuart flask rotator for 30 minutes at an appropriate speed.
Duration of treatment / exposure:
Treatment of the original parents was started on 9 October 1996 and was continuous throughout the study period of 70 days.
Frequency of treatment:
The rats were fed diets containing the test substance at the corresponding levels continuously until termination.
Details on study schedule:
Groups of 26 male and 26 female (F0 parents) weanling AIpk:APfSD (Wistar-derived) rats were fed diet containing 0 (control), 1000, 5000 or 20000ppm H112323. After 10 weeks, the animals were mated and allowed to rear the ensuing F1A litters to weaning.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 5000, 20000ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Group Dietary Concentration of HI 12323 (w/w) Identities of rats
males females
1 0 (Control) 1-26 105-130
2 1000 27-52 131-156
3 5000 53-78 157-182
4 20000 79-104 183-208

Control animals:
yes
Details on study design:
Justification for test system selection
The Alpk:APfSD strain of rat was used because of the substantial background data available for this strain, in the laboratory, relating to studies of this type.

The oral route was chosen for administration of HI 12323 as this represents the most appropriate route of human exposure based on the intended use of the substance and the known physical properties.

Dose level selection
The dose levels selected for this study were based on the results of previous studies in the same strain of rat carried out in this Laboratory and included an anticipated no-observed effect level and a level which achieves the limit dose for evaluation in this type of study 1000mg/kg/day.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
Clinical observations
Prior to the start of the study, all rats were examined to ensure that they were physically normal and exhibited normal activity. During the study all rats were observed daily for changes in clinical condition and behaviour and at the same time that bodyweight was recorded a detailed examination of each rat was made. Rats requiring euthanasia were killed and subjected to an examination post mortem Any rats found dead were subjected to an examination post mortem as soon as possible after death.

Bodyweights
Bodyweights: The duration of the pre-mating period was 10 weeks from the start of the study for the F0 parents.

The bodyweights of all rats were recorded at weekly intervals throughout the pre-mating periods

The initial weights for the F0 parents were recorded immediately before first feeding experimental diets

The female rats were weighed on days 1,8, 15 and 22 of gestation (the day on which sperm was detected in a vaginal smear was designated day 1 of gestation) and days 1,5,8, 11, 15 and 22 of lactation (the day of birth was designated day 1 of lactation), All rats were weighed at termination.

Food consumption
Food consumption for each cage of rats was recorded throughout the pre-mating period and calculated on a weekly basis. The food utilisation value per cage was calculated as the bodyweight gained by the rats in the cage per 100g of food eaten.
Oestrous cyclicity (parental animals):
Not measured.
Sperm parameters (parental animals):
Not measured.
Litter observations:
Clinical condition and survival
Litters were examined for dead or moribund pups at least once daily and any such pups were subjected to a gross examination post mortem abnormalities were recorded and the pups were discarded. A count of all live and dead pups was made within 24 hours of parturition (day 1) and thereafter at days 5, 8, 11, 15 and 22 post partum The sexes of the pups were also recorded at these times, Any clinical abnormalities seen in the pups were recorded

The following parameters were calculated for each litter:

% of pups live born = number of nups born live x 100
number of pups born live+number of pups born dead


% of pups surviving to day 22 = number of live pups on day 22 x 100
number of live pups on day I

Bodyweights
Individual pup bodyweights were recorded within 24 hours of birth (day 1) and at days 5, 8, 11,15 and 22 post partum Since pups were not individually identified, data were recorded by sex and litter.
Postmortem examinations (parental animals):
Termination
All surviving rats and those requiring euthanasia were killed by exsanguination under terminal anaesthesia induced by halothane Ph Eur , (FLUOTHANE, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) vapour

Organ weights
The following organs were removed trimmed and weighed from all rats surviving to scheduled termination:

Kidneys, testes, epididymides

Macroscopic examination
All animals were subjected to a mil examination post mortem which involved an external observation and a careful examination of all cranial, thoracic and abdominal organs and structures
The 10 males and 10 females with the lowest individual animal numbers from each group surviving to scheduled termination were selected for submission of an extended list of tissues.

The following tissues were examined in situ, removed and examined and fixed and stored in an appropriate fixative:

abnormal tissue; adrenal gland; aorta; bone femur (including joint); bone marrow - femur; brain (cerebrum, cerebellum and brainstem); caecum; cervix; colon; duodenum; epididymis; eye; Harderian gland*; heart; ileum; jejunum; kidney; liver; lung; lymph node - cervical; lymph node - mesenteric; mammary gland (females only); nasopharyngeal cavity*; nerve - sciatic; oesophagus; oral cavity*; ovary; pancreas; parathyroid gland; pituitary gland; prostate gland; rectum; salivary gland; seminal vesicle (including coagulating gland); skin; spinal cord (cervical, thoracic, lumbar); spleen; sternum; stomach; testis; thymus; thyroid gland; trachea; urinary bladder; uterus; voluntary muscle.

* indicates organs which were stored,


From all intercurrent deaths and all remaining animals the following tissues were submitted:

epididymis; kidney; mammary gland; ovary; pituitary gland; macro scopically abnormal tissue; prostate gland; seminal vesicle (including coagulating gland); testis; uterus; vagina.

Tissues from suspected infertile animals were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin , the kidneys of designated rats in intermediate groups were processed and examined histologically.
Postmortem examinations (offspring):
All surviving offspring were killed and discarded without further examination. All pups were killed on or soon after day 22 post partum.
Statistics:
Statistical methods included analysis of variance, analysis of covariance and Fisher's exact test.
Reproductive indices:
Not measured.
Offspring viability indices:
Not measured.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Blue colouration
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:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Clinical observations and mortality
The main treatment related finding was colouration of the rats by the test substance. Blue colouration was evident on the coat and skin and in the oral cavity of rats of all treatment groups, Some rats in the 20000ppm group also had blue eyes and a small number of rats in the 5000 and 20000ppm groups were also observed to have blue urine.

Several female rats were killed due to parturition difficulties or having failed to litter. These were from all dose groups and the finding was clearly unrelated to H112323 administration.

Bodyweight
There was no evidence of an effect of H112323 on the bodyweight of rats at dose levels up to and including 20000ppm.
There was no evidence of an adverse effect of H112323 on bodyweight during gestation or lactation.

Food consumption and utilisation
Food consumption was increased compared to controls in males and, to a lesser extent, in females receiving 20000ppm H112323. The increase was most evident at the start of the feeding period.
Food consumption was similar to control in rats receiving 1000 or 5000ppm H112323.
Food utilisation was less efficient in rats receiving 20000ppm H112323 than in controls. Food utilisation was similar to control in rats receiving 1000 or 5000ppm H112323.

Reproductive performance
The pre-coital interval was 1-4 days in the majority of pairings and there was no evidence of an effect of administration of H112323.
The length of gestation was not affected by administration of H112323.
The number of animals successfully mating was satisfactory in males and females in all groups and there was no evidence of an effect of H1123233.
The number of whole litter losses was 6, 2, 3 and 4 in the control, 1000, 5000 and 20000ppm dose groups respectively This number of whole litter losses is greater than expected for Alpk:APfSD rats but is clearly not related to treatment.
The percentage of pups live born was 97% or greater in all groups and was unaffected by administration of H112323.

Organ weights
Kidney weights were increased in both sexes receiving 20000 and 5000ppm. However the difference from control was only statistically significant in females at the 5000ppra dose level Testes weights were slightly increased in males receiving 20000ppm, There was no evidence of an effect on epididymis weight in any dose group.

Macroscopic findings
A number of rats died or were killed intercurrently. The findings were varied and apart from test substance colouration were not clearly treatment related.
There was a dose related blue discolouration of a wide variety of tissues with the majority of animals affected at 20000ppm and a lesser number at 5000ppm. Intestinal contents and the tail, limb and other skin sites were also discoloured in some rats at 1000ppm.
In addition there was a dose related increased incidence of dark kidneys at all dose groups in both sexes and a dose related increased incidence of dark spleens in males at all dose groups.

Microscopic findings
At termination there was a treatment related increase in the severity of tubular basophilia in the kidneys, Slight or moderate lesions were present in males at 20000 and females at 20000 and 5000ppm. Although minimal degrees of this change were not present in the designated control rats at termination an incidence of one rat from the infertile group was observed in males. The single incidence of minimal tubular basophilia observed in a female at 1000ppm is therefore considered not to be treatment related.
There were no other treatment related findings and no findings which correlated with the macroscopically observed blue discolouration or dark appearance of internal organs.

Microscopic findings in infertile animals
Of the rats considered to be infertile or suspected infertile, a number of findings were present to explain the infertility in some rats including dystocia, endometritis, vaginitis, or imperforate vagina. However in the majority of rats no pathological change was observed to explain the reproductive failure. There were no treatment related findings observed in these rats with the possible exception of two female rats dosed at 20000ppm with slight tubular basophilia.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Blue colouration
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Clinical observations
The only treatment related clinical observations were attributable to colouration or staining of the test substance. This was evident in the nostrils and on the skin or coat of the majority of offspring.

Bodyweight
Male and female pup weights were similar in control and treated groups.
The higher total litter weight in the 1000ppm group reflects the increased litter size in this group and is considered not to be related to treatment.

Investigations postmortem - offspring
Apart from colouration by the test substance there was no evidence of treatment related findings in those pups which were found dead or killed for humane reasons and were examined macroscopically.

Effect levels (F1)

Dose descriptor:
dose level:
Generation:
F1
Effect level:
> 20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effect was observed in the reproductive system at dietary levels. Dose level equates to 1000 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No treatment related effect was observed in the reproductive system at dietary levels up to and including 20000ppm of the test substance.

The target organ was the kidney and 1000ppm was a no-effect level for pathological change.
Executive summary:

Study conducted to OECD guideline reference 415 ; One-Generation Reproduction Toxicity Study and Annex V to Council Directive 67/548/EEC (One-generation reproduction toxicity test). Study in accordance with the Principles of Good Laboratory Practice (GLP).

No treatment related effect was observed in the reproductive system at dietary levels up to and including 20000ppm of the test substance.

 

The target organ was the kidney and 1000ppm was a no-effect level for pathological change.