Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

Diquat dibromide

EC number: 201-579-4 | CAS number: 85-00-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Toxic effect type:: concentration-driven

Effects on fertility

Description of key information

- The NOAEL for parental systemic toxicity was 2.80 mg/kg/day and for reproductive toxicity was 64.82 mg/kg/day of test substance in rats; similar to OECD 443; Hodge 1990

Link to relevant study records

Reference

Endpoint:: two-generation reproductive toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 07 Dec 1987 to Oct 1988
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: comparable to guideline study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:: equivalent or similar to guideline
Guideline:: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:: equivalent or similar to guideline
Guideline:: EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Principles of method if other than guideline:: The following were not evaluated: oestrus cycle length, sperm parameters (motility and morphology), the age and body weight at vaginal opening or balano-preputial separation of F1 offspring, a vaginal smear at necropsy to determine the stage of the oestrus cycle. The following organ weights were not recorded for P and F1 animals: uterus (with oviducts and cervix), ovaries, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands and their fluids (as one unit), brain, liver, kidneys, spleen, pituitary, thyroid and adrenal glands. Brain, spleen and thymus weights of F1 and F2 pups (one randomly selected pup/sex/litter) were not recorded. Full histopathology of vagina, uterus with cervix, ovaries, one testis and one epididymis, seminal vesicles, coagulating gland and target organs from parental animals control and high dose was not performed (including detailed testicular and epididymal histopathological examination and quantitative evaluation of primordial follicles for F1 females). Full histopathology of abnormal tissue and target organs from pups with external abnormalities or clinical signs as well as organs of the reproductive system from one pup/sex/litter, F1 and F2 was not conducted. The dose interval was 5 fold in this study (rather than no more than 3 fold) although effect and no effect levels have been demonstrated. Daily dosing of the parental (P) animals began when they were approximately 4 weeks old and continued for 12 weeks prior to mating (rather than 5-9 weeks of age at the start and continuing for at least 10 weeks prior to the mating period). A 3 week mating period was used. Rationale for dose level selection was not reported. Dose achieved for P and F1 animals is reported for pre-mating periods only. These deviations from the current regulatory guideline listed above are considered not to compromise the scientific validity of the study.
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Species:: rat
Strain:: other: Alpk:APfSD (Wistar-derived)
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Age at study initiation: (P) 28 days
- Mean weight at study initiation: (P) Males: 85 g; Females: 80 g; (F1) Males: 120 g; Females: 110 g
- Housing: on arrival two per cage, sexes separately. During paring one female was housed with one male. After mating all parent animals were individually housed. Animals were housed in multiple rat racks. The cages had solid stainless steel sides; the floor, back and front were constructed of 14 standard wire gauge stainless steel mesh at 1.27cm centres. The cages were suspended over collecting trays lined with absorbent paper. A glass jar containing the appropriate diet was placed in each cage.
- Diet: CT1 diet, ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-25
- Humidity (%): 37-82
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 Dec 1987 To: Oct 1988
Route of administration:: oral: feed
Vehicle:: water
Details on exposure:: DIET PREPARATION
The appropriate aliquot of the test substance was mixed with 25 mL of deionised water and added to 500 g of diet to prepare a premix. This was mixed using a pestle and mortar. The compound bottle was washed out with 2 x 25 mL of deionised water which was added to the mixture. The mixture was then passed through a 1.0 mm sieve and any lumps ground down using a pestle and mortar. Control premixes were prepared similarly but without the addition of the test substance. The premix was mixed in for 10 minutes after which 2 x 250 g of diet were added at 5 minute intervals to give a 1 kg dry premix. The 1 kg premix was added to 59 kg diet and mixed.
Diets were dispensed into glass jars of 350 mL capacity fitted with a stainless steel follower held in place with a lid. Trays of diet jars were stored in the diet storage area and fed to the rats as required.
Details on mating procedure:: - M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear

MATING PROCEDURE
The females from the P0 and F1 generations were mated with males from the same group after the premating period. They were allowed to rear the resulting litters to weaning. Brother-sister matings were avoided in both generations.
During mating one female was housed with one male from the adjacent cage. Vaginal smears were examined daily to determine when mating occurred (as shown by the presence of sperm). After 21 days if there was no evidence of mating, the first male was removed and after a three day interval a second male from the same group and which had successfully produced a positive vaginal smear with another female was introduced into the cage. The period between the two matings ensured that the paternity of any litter produced could be unequivocally ascribed. No further pairing was made if the second pairing was unsuccessful.

When any female showed a positive vaginal smear, the male was removed and the female was individually housed. Pregnancy was presumed when abdominal enlargement and weight gain were seen. All females with sperm-positive smears which failed to show signs of pregnancy were remated generally 14 days after the smear was recorded as described above (for females for which there was no evidence of mating). At approximately day 15 of pregnancy the cages housing the females were fitted with solid floors and supplied with autoclaved paper bedding material. Water bottles were also fitted to the cages and the automatic watering system disconnected at this time. The females remained in these cages throughout gestation and lactation.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Samples were taken from the first batches of all dietary levels and analysed by a fully quantitative analysis method to ensure that they contained the test substance at the inclusion rate specified. Further samples were taken for full quantitative analysis at approximately 8 week intervals.

The mean concentrations for all batches of diet preparations analysed were typically within 10% of the nominal concentration.
The homogeneity of the test substance in CT1 diet was confirmed by analysing samples from the 16 ppm and 400 ppm diets. The test substance has been shown to be stable in CT1 diet for approximately 6 weeks in previous studies.
The test substance had been shown to be stable in CT1 diet for approximately 6 weeks in previous studies.
Duration of treatment / exposure:: Twelve (or 11 for F1) weeks prior to mating, during mating, gestation and lactation.
Frequency of treatment:: Continuous
Details on study schedule:: - F1 parental animals not mated until 11 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 36 days of age.
Dose / conc.:: 16 ppm
Remarks:: Group 2; equivalent to 1.7, 1.6 and 1.5 mg test substance ion/kg bw in F0/F1 females, F0 males and F1 males, respectively.
Dose / conc.:: 80 ppm
Remarks:: Group 3; equivalent to 8.4, 7.9 and 7.8 mg test substance ion/kg bw/day in F0/F1 females, F0 males and F1 males, respectively.
Dose / conc.:: 240 ppm
Remarks:: Group 4; equivalent to 40.4, 38.7, 35.7 and 34.7 mg test substance ion/kg bw/day in F0 females, F0 males, F1 females and F1 males, respectively
No. of animals per sex per dose:: 30
Control animals:: yes, plain diet
Details on study design:: PARENT SELECTION
- 30 males and 30 females per group were selected from the F1a litter to become the next generation (F1) parents.
- The rats were 36 days of age at selection. The difference in age of offspring at selection was minimised by selecting only from litters which had reached day 11 when the procedure was planned and selection was from 20 litters per group except for the 16 ppm group where it was from 16 litters.
- The F1a and F2A litters were separated from the mothers at day 22 post partum but remained housed by litter until selection at day 36 post partum.
- After selection, the premating and breeding was repeated until the F2A litter had been produced.
- Litters containing 6 to 18 pups at Day 11 were considered suitable for selection; maximum of two males and two females selected from any litter; not all litters resulting in remates were included so that rats selected for the next generation were as close in age as possible.

ALLOCATION OF ANIMALS
The allocation of animals followed these critera:
- animals within a cage were not closely related
- the two males and two females in adjacent cages (same group and replicate) were not related
- animals within a cage were as close in age as possible
Positive control:: None
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS
- Time schedule: daily
- Moribund or dead animals were sent for post mortem examination.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: daily
- A detailed examination was made at the same time it was weighed. An additional weekly examination of the mouths using a light source and a probe to depress the tongue of all F1 rats was instituted from approximately 4 weeks after selection. Day 22 pregnant and day 1 lactating females were exempt from the additional examination.

BODY WEIGHT
- Time schedule during premating period: weekly intervals
- After the premating period the male rats were weighed approximately once per month until termination in order to monitor their health status.
- Female rats were weighed on days 1, 8, 15 and 22 of pregnancy (the day on which sperm were detected in a vaginal smear was designated day 1 of pregnancy). They were also weighed on days 1, 5, 11, 16, and 22 of lactation. All animals were weighed at termination.

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

OPTHALMOSCOPY
Prior to the start of the study the eyes of 15 male and 15 female F0 rats per group; during week 12 the eyes of all adult F0 animals in groups 1 and 4, and prior to termination (approximately 24 weeks) the eyes of all F0 animals were examined. The eyes of all F1 adult rats were examined approximately 4 weeks after selection, at the end of the premating period (approximately 11 weeks) and prior to termination (approximately 21 weeks). Examination was performed using a Fison's binocular indirect ophthalmoscope after the instillation of a mydriatic into the eyes to dilate the pupil.

REPRODUCTIVE PARAMETERS
- The fertility of each male and female was established by the success of each mating. The criterion for determining the fertility was the production of a viable litter i.e. a litter in which at least one pup was found live at day 1.
- Length of gestation was measured in days from date of positive smear to date of birth of a viable litter.
- Precoital interval, the time in days between the date of pairing and the date of positive smear, for all pairings which resulted in a positive smear.
Oestrous cyclicity (parental animals):: Not examined
Sperm parameters (parental animals):: Not examined
Litter observations:: PARAMETERS EXAMINED
The following litter observations were made. Each pup was individually assessed as follows; as pups were not individually identified, data were recorded by sex and litte.
- Viability/mortality: Determined as viable or stillborn within 24 hours of parturition (day 1) and thereafter checked for viability at days 5, 11, 16, 22, 29 and 36 post partum.
- Sex: Determined by external examination at the same time as the viability check.
- Clinical signs: Recorded at the same time as the viability check. The mouths of all pups from 5 control and 240 ppm F2A litters were examined for mouth lesions on days 11, 16, 22, 29 and 36 post partum.
- Body weight: Individual pup body weights recorded within 24 hours of birth (day 1) and on days 5, 11, 16, 22, 29 and 36 post partum. Litters were separated from their mothers at day 29 post partum. Terminal body weights of pups selected for full examination post mortem were recorded. The F1 and F2 pups selected at day 36 post partum were weighed at this time, to give the initial parent body weight values, and thereafter on the same day of each week for the duration of the premating period.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: All F0 and F1 males producing viable litters were killed shortly after completion of mating and consideration of litter data
- Female animals: All females parents from each generation were killed at approximately the same time after weaning their litters. Any female apparently pregnant but failing to produce a litter was killed on or soon after the expected day 25 of pregnancy

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- The number of implanations sites in each uterine horn was recorded for each mated female
- The tissues were prepared for microscopic examination: macroscopically abnormal tissues, eye, kidney, head including tongue, reproductive tract (testis, epididymis, prostate, seminal vesicles from males and uterus, cervix/vagina, ovary, mammary gland from females). All submitted tissues were examined from rats killed or dying intercurrently.
- Testes and kidneys were weighed
- The reproductive tract was examined histologically from all F0 and F1 adults which were suspected to be infertile
- Macroscopically abnormal tissues, eye, kidney and tongue were examined from adults from the control and top dose group. Macroscopically abnormal tissues and tongue were examined from the other dose groups.
- In addition, oral cavity (for hard palate) was examined from all F1 adults.
- Microscopic examination: Tissues for histological examination were routinely processed, embedded in paraffin wax and 5 µm thick sections were cut and stained with haematoxylin and eosin.
Postmortem examinations (offspring):: SACRIFICE
- Moribund offspring and all offspring surviving to termination were sacrificed.
- Pups remaining after selection from F1a and F2a litters were killed at or soon after day 36 post partum.
- Five F1a and ten F2a pups from each sex and group were selected at random (not more than one pup per sex per litter) for full post mortem examination.

GROSS NECROPSY
- Any pups (up to and including 18 days of age) found dead or with behavioural, functional, or morphological abnormalities were examined post mortem by dissection.
- Pups over 18 days of age found either dead or moribund were subjected to a full post mortem examination.
- After selection of pups for continuation to the next generation (where appropriate) and those for full examination post mortem, all remaining pups showing significant clinical abnormalities and where possible two of the remaining clinically normal pups of each sex from each litter, also received a gross necropsy where macroscopically abnormal tissues only were preserved.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Macroscopically abnormal tissues, eye, kidney, tongue, oral cavity for hard palate and reproductive tract were preserved in fixative.
- Kidneys and testes were weighed from pups designated for full post mortem examination.
Statistics:: See "Any other information on materials and methods incl. tables" section
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: A variety of clinical signs was recorded, some of which were related to administration of the test substance. Opaque eye(s) were observed in the majority of F0 rats receiving 400 ppm test substance, the first instances being seen at week 12 in females and at week 13 in males.
Tray paper staining and the related finding of coloured urine were seen in F0 males receiving 400 ppm. There was a low incidence of one or more of these findings in F0 males receiving 80 ppm.
F0 rats of both sexes receiving 400 ppm had piloerection and males showed lack of grooming.
Dermal irritation (if dermal study):: not examined
Mortality:: mortality observed, non-treatment-related
Description (incidence):: All F0 males survived to the scheduled termination but a single F0 female dosed at 16 ppm was killed in extremis. Its clinical signs included a swollen neck and pathological examination showed it had a lymphosarcoma.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: - Body weight gain was reduced in F0 males and females receiving 400 ppm test substance. At the end of the premating period body weight gains were 15 and 10% less in males and females respectively than those of the corresponding controls. There was no effect on body weight gain in F0 rats receiving 16 or 80 ppm doses.
- Pregnancy weights and pregnancy weight gains were lower than those of controls in F0 females receiving 400 ppm. There was no effect on pregnancy weight gain in F0 females receiving 16 or 80 ppm doses.
- Lactation weights and weight gains were lower than in controls in F0 rats receiving 400/240 ppm test substance. There was no effect on lactation weight or weight gain in F0 rats receiving 16 or 80 ppm.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: - Food consumption was lower than that of controls in F0 rats receiving 400 ppm test substance, particularly at the beginning of the dosing period. No effects were apparent in F0 at the 16 or 80 ppm dose levels.
- Food consumption during pregnancy was lower than that of controls in F0 rats receiving 400 ppm test substance. No effects were apparent in F0 rats receiving 16 or 80 ppm dose levels.
- The pattern of food consumption during lactation was similar to that during pregnancy.
- Compound intake: The pattern of dose received in mg test substance/kg bw/day was as expected. It was highest at the start of the study/selection when young rats were growing rapidly, and gradually levelled offs the study progressed.
Food efficiency:: no effects observed
Description (incidence and severity):: Food utilisation was not adversely affected in F0 rats.
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: - At the end of the premating period there was an increased incidence of partial cataracts and a few total cataracts in males and females treated with 400 ppm test substance when compared with controls. Females were slightly more severely affected than males.
- At pre-termination the severity of the changes at 400 ppm had progressed with more rats showing total cataracts. All rats treated with 400 ppm had cataracts. At 24 weeks no cataracts were seen in rats fed 16 ppm test substance and, only single incidences were apparent in males in the control and 80 ppm groups.
- The nature of the test substance induced cataracts varied from animal to animal and also varied according to the time period of examination. The less severe cataracts (partial) were characterised by the following features: increased transparency or clearing of the lens (this was believed to result from a change in optical density or loss of lines of discontinuity rather than opacity), focal areas of opacity (usually posterior) with or without increased demarcation of the posterior suture lines, vacuoles (usually posterior). The more severe cataracts (total) were characterised either by opacity of the whole or greater part of the lens or by shrinkage of the lens with opacification. The cataracts tended to be bilateral although the degree of change in each eye was not necessarily the same. The severity and incidence of cataracts increased with time and females were more severely affected than males.
- With the exception of the changes associated with cataracts there were no other treatment-related ocular effects.
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Endocrine findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: - Ocular opacity observed in animals receiving 400 ppm was shown histologically to be due to cataractous change. All rats in this group
showed bilateral cataractous change graded as marked or severe in the majority of animals and many showed secondary ocular changes eg retinal detachment and degeneration, vitreous adhesions, posterior synechiae and lens-induced inflammation. Cataract formation and associated changes were not observed in concurrent control animals.
- Ulceration of the tongue was confirmed histologically in 7 males and 25 females receiving 400 ppm test substance. Eight other males at this dose level showed one or more of the following changes in the tongue: loss of normal arrangements of epithelial papillae and rete pegs, abscess formation, granulomatous inflammation, mixed inflammatory cell infiltration or acanthosis. Slight ulceration of the tongue was seen in one female receiving 80 ppm test substance. No abnormality of the tongue was observed in any animal receiving 16 ppm or in concurrent controls.
- An area of discolouration of the hard palate observed in the fixed tissue at trimming in one male receiving 400 ppm test substance was shown histologically to be due to ulceration.
Histopathological findings: neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: One F0 female dosed at 16 ppm was killed in extremis. Its clinical signs included a swollen neck and pathological examination showed it had a lymphosarcoma.
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, non-treatment-related
Description (incidence and severity):: - Pre-coital interval, gestation length, proportion of pups born live and proportion of pups surviving to weaning, and the total number of litters produced were not affected by the administration of test substance.
- The number of whole F1a litter losses was 1, 2, 1 and 4 in the control, 16, 80 and 400ppm groups respectively. One of the losses at the 400ppm dose level was due to accidental early termination of the dam. Litter size from birth was slightly decreased in the F1a litter of the 400 ppm group but no similar effect was observed in the F2a litter where the top dose was 240ppm.
- One F0 female receiving 16 ppm test substance failed to produce a litter. Ovarian atrophy was the probable reason for infertility in this animal.
- Some (up to 7) males in all groups failed to sire any offspring, but the incidence of infertility was unrelated to treatment. Slight testicular tubular degeneration was detected in one control animal. No abnormalities were detected in any other male suspected of infertility.
Dose descriptor:: NOEL
Remarks:: Systemic toxicity
Effect level:: 3.18 mg/kg bw/day
Based on:: other: pure test substance
Sex:: female
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
: Key result
Dose descriptor:: NOEL
Remarks:: Systemic toxicity
Effect level:: 2.98 mg/kg bw/day
Based on:: other: pure test substance
Sex:: male
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOEL
Remarks:: Systemic toxicity
Effect level:: 16 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Original value presented in study. Dietary equivalent to 1.6 and 1.7 mg test substance ion/kg bw/day for males and females, respectively
: Key result
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 72.29 mg/kg bw/day
Based on:: other: pure test substance
Sex:: male
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 75.47 mg/kg bw/day
Based on:: other: pure test substance
Sex:: female
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 240 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Original value presented in study. Dietary equivalent to 38.7 and 40.4 mg test substance ion/kg bw/day mg test substance/kg bw/day for males an females, respectively.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 2.98 other: mg/kg bw/day. Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
System:: eye
Organ:: lens
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: The incidence of opaque eyes in F1 rats receiving 240 ppm test substance cation was similar to that in the previous generation for females and slightly higher for males. The observation was first recorded during week 10 (females) or week 11 (males) after selection of the F1. Opaque eyes were not evident in controls or in rats receiving 16 or 80 ppm dose.
Ulceration of the palate and/or tongue was observed in F1 males and females receiving 240 ppm test substance cation from week 4.
There was a low incidence of tray paper staining and the related finding of coloured urine in F1 males receiving 80 ppm. There was no evidence of tray paper staining or coloured urine in either sex of F1 receiving 240 ppm or in either generation of the control or 16 ppm group.
After selection, F1 rats at the top dose level were observed to be ungroomed.
Dermal irritation (if dermal study):: not examined
Mortality:: mortality observed, non-treatment-related
Description (incidence):: One F1 male receiving 240 ppm died in week 9. It had malocclusion. Two F1 females were found dead: one control in week 3 (which was found to have cystitis) and one from the 240 ppm group in week 6 (which had unilateral hydronephrosis). An F1 female from the 16 ppm group was killed for investigation after showing weight gain but failing to litter and was found to have an obstruction in the cervix. An F1 female from the 240 ppm group was killed as it had an imperforate vagina and could not be paired. Imperforate vagina is a congenital abnormality seen occasionally in the Alpk:APfSD rat.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: - Body weights for F1 receiving 400 ppm were much lower than for controls at selection. These rats failed to grow satisfactorily and concerns about the viability of these rats led to the decision to decrease the top dose from 400 to 240 ppm at approximately week 4 after selection. After the change of dose level the rats showed similar weight gains to controls although weights at the end of the premating period were less than those of controls. There was no effect on body weight in rats receiving 16 or 80 ppm test substance.
- Pregnancy weights were lower than in controls but weight gains were not affected in F1 females receiving 240 ppm. There was no effect on pregnancy weight gain in F1 females receiving 16 or 80 ppm test substance.
- Lactation weights and weight gains were lower than in controls in F1 rats receiving 240 ppm test substance cation. There was no effect on lactation weight or weight gain in F1 rats receiving 16 or 80 ppm.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: - Food consumption was markedly lower in F1 rats of the top dose group than in controls to weeks 3/4 after selection. However, once the dose was decreased food consumption was only marginally lower than that of controls. No effects were apparent in F1 rats at the 16 or 80 ppm dose levels.
- No effects were apparent on food consumption during pregnancy and lactation in rats at any dose level.
- Compound intake: The pattern of dose received in mg test substance/kg bw/day was as expected. It was highest at the start of the study/selection when young rats were growing rapidly, and gradually levelled offs the study progressed. There was a marked reduction in dose received for F1 rats receiving 400 ppm until the dose was amend to 240 ppm.
Food efficiency:: effects observed, non-treatment-related
Description (incidence and severity):: The efficiency of food utilisation in F1 rats receiving 400/240 ppm test substance ion was greater than that of controls. A slight increase in food utilisation was observed in F1 females receiving 80 ppm but this was considered incidental to treatment as there was no similar effect in males nor in the F0 females.
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: At 4 weeks after selection there was an increased incidence of partial cataracts in males and females treated with 240ppm test substance cation with the majority of animals affected.
At premating the lesions had progressed such that the incidence of cataracts was greater and more cataracts were total in nature. At 240 ppm all rats were affected, females were more severely affected than males. In females at 80 ppm there was a marginal increase in rats with partial cataract compared to controls.
One female rat at 240 ppm had iritis, probably occuring as a complication of cataract.
At pre-termination the lesions had progressed such that most animals treated with 240 ppm test substance cation had total cataracts and more rats (both male and female) showed iritis. In addition, there was a small increase in the incidence of partial cataract in females at 80 ppm.
Although one male and one female at 16 ppm had a partial cataract this was considered not to be of toxicological significance as a similar incidence was found in the control group.
The nature of the test substance induced cataracts varied from animal to animal and also varied according to the time period of examination. The less severe cataracts (partial) were characterised by the following features: increased transparency or clearing of the lens (this was believed to result from a change in optical density or loss of lines of discontinuity rather than opacity), focal areas of opacity (usually posterior) with or without increased demarcation of the posterior suture lines, vacuoles (usually posterior). The more severe cataracts (total) were characterised either by opacity of the whole or greater part of the lens or by shrinkage of the lens with opacification. The cataracts tended to be bilateral although the degree of change in each eye was not necessarily the same. The severity and incidence of cataracts increased with time and females were more severely affected than males.
There were no other treatment-related ocular effects.
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Endocrine findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: Absolute kidney weights were slightly lower than controls in F1 animals receiving 240 ppm, although the change was not evident after correction for bodyweight.
Testes weights were not affected by test substance administration.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: Ocular opacity was observed in 27/30 animals of each sex at termination at the reduced top dose level of 240 ppm test substance cation. Ulceration of the hard palate in animals receiving 240 ppm, was observed in 4/30 males, 2/30 females. Many animals (17/30 males, 15/30 females) showed loss of palatine ridges; this observation being suggestive of healed ulceration. Ulceration of the tongue was also seen in 12 females receiving 240 ppm, and in 1 at the 80ppm dose level, but not in any male animal.
Caecal enlargement was not observed in any adult animal at termination.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: Cataractous change was observed at a similar incidence and severity as that recorded in F0 adults. All animals receiving 240 ppm except one female were affected and in the majority the lesion was bilateral, marked or severe and accompanied by secondary intraocular changes.
Ulceration of the hard palate was confirmed histologically in 5 males and 2 females receiving 240 ppm test substance cation. A greater number of animals (3 males, 15 females) in this group showed changes characteristic of healed ulceration. In most of these the palate mucosa showed loss of normal arrangement of epithelial ridges and rete pegs. A few showed other changes such as acanthosis, hyperkeratosis, granulomatous inflammation and mixed inflammatory cell infiltration. Ulceration of the tongue was not observed histologically in F1 adults but 2 males and 11 females in the 240 ppm dose groups and one female receiving 80ppm showed changes characteristic of repair after ulceration, similar to those observed in the hard palate. Loss of normal arrangement of papillae and rete pegs was the most frequently recorded change. These changes in the tongue and hard palate were not observed in any animal in the control or 16 ppm dose group.
A slightly increased incidence of minimal hypertrophy and hyperplasia of collecting duct epithelium and minimal or slight tubular dilatation in the renal papilla was observed in the kidneys of animals of both sex receiving 240 ppm test substance.
Histopathological findings: neoplastic:: no effects observed
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: no effects observed
Description (incidence and severity):: Pre-coital interval, gestation length, proportion of pups born live and proportion of pups surviving to weaning, and the total number of litters produced were not affected by the administration of test substance.
There was no evidence of an effect of treatment on total litter losses.
The slightly increased litter size in the F2A litters at 16 ppm test substance was considered not to be related to treatment as there was no effect at the higher doses.
The incidence of infertile animals was low and unrelated to treatment. None of the few changes detected in the reproductive tract of these animals was considered to be related to treatment.
: Key result
Dose descriptor:: NOAEL
Remarks:: Systemic toxicity
Effect level:: 2.8 mg/kg bw/day
Based on:: other: pure test material
Sex:: male
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Systemic toxicity
Effect level:: 3.18 mg/kg bw/day
Based on:: other: pure test substance
Sex:: female
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Systemic toxicity
Effect level:: 16 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: body weight and weight gain; gross pathology
Remarks on result:: other: Original value in presented in study. Dietary equivalent to 1.5 and 1.7 mg test substance ion/kg bw/day for males and females, respectively
: Key result
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 64.82 mg/kg bw/day
Based on:: other: pure test substance
Sex:: male
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 66.68 mg/kg bw/day
Based on:: other: pure test substance
Sex:: female
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Remarks:: Reproductive toxicity
Effect level:: >= 240 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: reproductive performance
Remarks on result:: not determinable due to absence of adverse toxic effects
Remarks:: Original value in presented in study. Dietary equivalent to 34.7 and 35.7 mg test substance ion/kg bw/day for males and females respectively.
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 2.8 other: mg/kg bw/day. Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
System:: eye
Organ:: lens
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: A variety of clinical observations was recorded, the majority of these were of a type and incidence expected for the Alpk:APfSD strain of rat and unrelated to treatment. However, the incidence of signs of urinary incontinence, distended abdomen and piloerection were increased in F1a litter of the top dose group.
Dermal irritation (if dermal study):: not examined
Mortality / viability:: no mortality observed
Description (incidence and severity):: The proportion of pups live born and the proportion of pups surviving to weaning were not affected by test substance.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Pup weight gain was lower in the top dose in both generations. A statistically significant decrease in total litter weight was apparent for all time points except day 5 for the F1a litters with a marked difference from day 22 onwards. The slightly lower values for pup weight gain in F1a litters of the 80 ppm group are considered not to be related to the test substance as no similar effect was seen in the F2a litters and total litter weight was not affected.
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: no effects observed
Description (incidence and severity):: Ocular opacity was observed in 1 control female pup.
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: not examined
Nipple retention in male pups:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: - Absolute kidney weights were lower than controls in F1a offspring receiving 400 ppm test substance although the effect was not evident after correction for bodyweight.
- Testes weights were not affected by the test substance administration.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: In pups up to 18 days of age no specific cause (s) of death was established. The main pathological finding in decedent pups (18 days or older) was inflammatory disease of the urinary tract (cystitis, pyelitis, pyelonephritis); 6 pups were affected including 3 controls.
- Ulceration of the hard palate was observed in 22/56 male and 19/51 female pups from 18 litters receiving 400 ppm test substance. Ulceration of the tongue was not seen in treated pups of this generation. Ocular opacity was observed in 1 control female pup.
The caecum was distended and/or gas filled in 8 males and 7 females from 8 litters receiving 400 ppm.
Unilateral or bilateral ureteric dilatation, accompanied by enlargement of one or both kidneys due to pelvic dilatation, was recorded in 4 males and 3 females from 4 litters receiving 400 ppm test substance.
Histopathological findings:: effects observed, treatment-related
Description (incidence and severity):: Marked or severe ulceration of the hard palate was confirmed microscopically in 21 males and 19 females from 17 litters receiving 400 ppm test substance cation. This lesion was not observed in any animal from the control or two lower dose groups. Ulceration of the tongue was not observed and all tongues examined from animals in control and treated groups were within normal limits.
A few changes were observed in the urinary tract which were confined to or occurred at a slightly increased incidence in pups receiving 400 ppm test substance cation. Minimal to moderate cortical tubular dilatation was observed in 13 males and 8 female pups from 9 litters receiving 400 ppm. In one of these males and 3 females the kidney also showed minimal or slight multifocal balloon degeneration of cortical tubular epithelium. Slight cortical tubular dilatation was seen in one control pup and one receiving 16ppm. Pyelitis was observed in 5 males and 3 females from 6 litters receiving 400 ppm test substance cation. This lesion was also recorded in 3 control female litter mates and one female receiving 80 ppm. The control females also showed marked or severe cystitis. Marked pyelonephritis was also seen in two unrelated females receiving 80 ppm test substance. Minimal or marked papillary necrosis was seen in 2 males receiving 400 ppm. Moderate or marked ureteritis was observed in 3 males and 4 females from 4 litters receiving 400 ppm and in one of the females receiving 80 ppm which showed marked pyelonephritis. Three of the animals receiving 400ppm also showed marked cystitis. An increased incidence of minimal hypertrophy and hyperplasia of collecting duct epithelium was observed in males receiving 80 ppm compared with other control and treated groups. Individual pups often showed several changes affecting different regions of the urinary tract. Several pups from the same litter were often affected and the number of pups showing one or more of these changes was increased in pups receiving 400ppm, especially in males.
Cataract formation was not seen in the lens of any control or treated F1 pup.
Other effects:: not examined
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 15.69 mg/kg bw/day
Based on:: other: pure test substance
Sex:: male/female
Basis for effect level:: clinical signs; gross pathology
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 80 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: clinical signs; gross pathology
Remarks on result:: other: Original value in presented in study. Dietary equivalent to 8.4 mg test substance ion/kg bw/day for maternal animals.
Critical effects observed:: yes
Lowest effective dose / conc.:: 15.69 other: mg/kg bw/day. Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: A variety of clinical observations was recorded, the majority of these were of a type and incidence expected for the Alpk:APfSD strain of rat and unrelated to treatment. However, the incidence of signs of urinary incontinence was increased in F2a litters of the top dose group.
Dermal irritation (if dermal study):: not examined
Mortality / viability:: no mortality observed
Description (incidence and severity):: The proportion of pups live born and the proportion of pups surviving to weaning were not affected by test substance.
Body weight and weight changes:: no effects observed
Description (incidence and severity):: Pup weight gain was lower in the top dose in both generations. A statistically significant decrease in total litter weight was apparent for all time points. There were small differences only for the F2a litters from day 16 onwards and these did not achieve statistical significance. The slightly lower gains in F2a litters of the 16 ppm group reflect the increased litter size and are not attributable to treatment.
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: effects observed, treatment-related
Description (incidence and severity):: Ocular opacity was observed in 1 male receiving 80 ppm and 1 female receiving 240 ppm test substance cation species.
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: not examined
Nipple retention in male pups:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: Testes weights were not affected by test substance administration.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: - Ocular opacity was observed in 1 male receiving 80 ppm and 1 female receiving 240 ppm test substance.
Caecal distension was recorded in 2 males and 2 females from the same litter receiving 240 ppm. Gross changes in the tongue or hard palate were not observed in any pups of this generation.
Histopathological findings:: effects observed, treatment-related
Description (incidence and severity):: Lesions of the lens and oral cavity were not observed in any F2 pup. Ocular opacity observed grossly in one pup in each of the 80 and 240 ppm groups was not due to cataractous change.
An increased incidence of minimal or slight hypertrophy and hyperplasia of collecting duct epithelium was observed in the kidneys of pups receiving 240 ppm test substance cation. The overall incidence in this group (ie in full and gross necropsy animals) was 55% of kidneys examined in males, and 41% in females compared with a background incidence range of 7-29% in other groups.
Marked cystitis was present in 5 male and female litter mates receiving 240 ppm test substance cation, and pyelitis or pyelonephritis was seen in 5 males and 5 females from 6 litters, also at this dose level. Neither of these changes was seen in any pup in control or lower dose group. One pup of each sex receiving 240 ppm from different litters showed slight cortical tubular dilatation. Minimal cortical tubular dilatation was seen in one control female.
The overall incidence of urinary tract lesions was slightly increased in pups receiving 240 ppm compared with control and lower dose groups as it was in the generation at the top dose level.
Other effects:: not examined
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Generation:: F2
Effect level:: 15.69 mg/kg bw/day
Based on:: other: pure test substance
Sex:: male/female
Basis for effect level:: clinical signs
Remarks on result:: other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation.
Dose descriptor:: NOAEL
Generation:: F2
Effect level:: 80 ppm
Based on:: other: test substance cation species
Sex:: male/female
Basis for effect level:: clinical signs
Remarks on result:: other: Original value in presented in study. Dietary equivalent to 8.4 mg test substance ion/kg bw/day for maternal animals
: Key result
Critical effects observed:: yes
Lowest effective dose / conc.:: 15.69 other: mg/kg bw/day. Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
System:: urinary
Organ:: kidney
Treatment related:: yes
Dose response relationship:: yes
Relevant for humans:: yes
: Key result
Reproductive effects observed:: yes
Lowest effective dose / conc.:: 15.69 mg/kg bw/day
Treatment related:: yes
Relation to other toxic effects:: reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:: no
Relevant for humans:: presumably yes
Reproductive effects observed:: yes
Lowest effective dose / conc.:: 80 ppm
Treatment related:: yes
Relation to other toxic effects:: reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:: no
Relevant for humans:: presumably yes
Conclusions:: At 400/240 ppm test substance cation there was evidence of toxicity in adults and offspring. There was a low incidence of toxicity at 80 ppm test substance cation in adults only. The no effect level for toxic effects in this study was 16 ppm test substance cation (2.80, 2.98 and 3.18 mg test substance/kg bw/day for F0 males, F1 males and F0/F1 females, respectively). The NOAEL for reproductive effects was 240 ppm test substance cation (72.29, 64.82, 75.47 and 66.68 mg test substance/kg bw/day for F0 males, F1 males, F0 females and F1 females, respectively).
Executive summary:: In a GLP compliant study, similar to OECD TG 416, groups of 30 male and 30 female (F0 parents) weanling Alpk:APfSD rats were fed diet containing 0 (control), 16, 80 or 400 ppm test substance cation. After twelve weeks the animals were mated and daily vaginal smear examinations were done to determine when mating occurred (designated day 1 of gestation). The ensuing F1A litters were reared to weaning. The breeding programme was repeated with the F1 parents selected from the F1A offspring that were mated at least 11 weeks after selection to produce the F2A litters. Due to adverse effects (ulceration of the hard palate) in the F1 rats receiving 400 ppm test substance cation, the dose was reduced to 240 ppm approximately 4 weeks after selection (9 weeks old). Test diets were fed continuously throughout the study. During the study all rats were observed daily for abnormalities in clinical condition and behaviour. Moribund or dead rats were examined post mortem. Parental food and body weights were measured throughout the study. Reproductive performance and selected offspring parameters were measured.Decreased body weight gains were observed in adults and pups receiving 400/240 ppm test substance cation. Signs of toxicity observed at 400/240 ppm included inflammatory lesions of the mouth, cataract in adults, piloerection and poor grooming and an increased incidence of pathological lesions of the kidney/urinary tract in pups. A slightly increased incidence of partial cataract in F1 females at 80 ppm test substance ion was seen, and single examples of the mouth lesion seen at the top dose level occurred in one female each in the F0 and F1 generations. There were no signs of toxicity in rats receiving 16 ppm test substance cation.A slight decrease in the number of pups per litter was seen in F1A litters at 400 ppm. No similar effects were seen at 240 ppm or in the F2A litters. At 400/240 ppm test substance ion there was evidence of toxicity in adults and offspring. There was a low incidence of toxicity at 80 ppm test substance ion in adults only. The no adverse effect level (NOAEL) for toxicity in this study was 16 ppm test substance ion (2.80, 2.98 and 3.18 mg test substance/kg bw/day for F0 males, F1 males and F0/F1 females, respectively). The NOAEL for reproductive effects was 240 ppm test substance ion (72.29, 64.82, 75.47 and 66.68 mg test substance/kg bw/day for F0 males, F1 males, F0 females and F1 females, respectively).

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 64.82 mg/kg bw/day
Study duration:: chronic
Species:: rat
Quality of whole database:: GLP compliant study, similar to OECD 443

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

Multigeneration study in the rat - OECD 443 (Hodge 1990)

In a GLP compliant study, similar to OECD TG 416, groups of 30 male and 30 female (F0 parents) weanling Alpk:APfSD rats were fed diet containing 0 (control), 16, 80 or 400 ppm test substance cation. After twelve weeks the animals were mated and daily vaginal smear examinations were done to determine when mating occurred (designated day 1 of gestation). The ensuing F1A litters were reared to weaning. The breeding programme was repeated with the F1 parents selected from the F1A offspring that were mated at least 11 weeks after selection to produce the F2A litters. Due to adverse effects (ulceration of the hard palate) in the F1 rats receiving 400 ppm test substance cation, the dose was reduced to 240 ppm approximately 4 weeks after selection (9 weeks old). Test diets were fed continuously throughout the study. During the study all rats were observed daily for abnormalities in clinical condition and behaviour. Moribund or dead rats were examinedpost mortem. Parental food and body weights were measured throughout the study. Reproductive performance and selected offspring parameters were measured.

Decreased body weight gains were observed in adults and pups receiving 400/240 ppm test substance cation. Signs of toxicity observed at 400/240 ppm included inflammatory lesions of the mouth, cataract in adults, piloerection and poor grooming and an increased incidence of pathological lesions of the kidney/urinary tract in pups. A slightly increased incidence of partial cataract in F1 females at 80 ppm test substance ion was seen, and single examples of the mouth lesion seen at the top dose level occurred in one female each in the F0 and F1 generations. There were no signs of toxicity in rats receiving 16 ppm test substance cation. A slight decrease in the number of pups per litter was seen in F1A litters at 400 ppm. No similar effects were seen at 240 ppm or in the F2A litters. At 400/240 ppm test substance ion there was evidence of toxicity in adults and offspring. There was a low incidence of toxicity at 80 ppm test substance cation in adults only. The no adverse effect level (NOAEL) for toxicity in this study was 16 ppm test substance cation, equivalent to 2.80, 2.98 and 3.18 mg pure test substance/kg bw/day for F0/F1 males and females, respectively. The NOAEL for reproductive effects was 240 ppm test substance ion, corresponding to 72.29, 64.82, 75.47 and 66.68 mg pure test substance/kg bw/day for F0/F1 males and and females, respectively.

Effects on developmental toxicity

Description of key information

- The NOAEL in rabbits for maternal toxicity was 1.9 mg/kg bw/day and for developmental toxicity was 5.64 mg/kg bw/day pure test substance (Hodge 1989)

- The NOAEL in rats for maternal toxicity was 7.47 mg/kg bw/day and for developmental toxicity was 22.4 mg/kg/day pure test substance (Wickramaratne 1989)

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Apr 1988 to 03 Jun 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: not reported
- Weight at study initiation: 3.11 - 4.03 kg
- Housing: Individually housed in mobile rabbit units
- Diet: CRB pellets, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 6 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 46 - 66
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From 25 Apr 1988 To: 03 Jun 1988

Route of administration:

oral: gavage

Vehicle:

other: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was formulated in deionised water. The concentration of substance was adjusted to give a constant volume of 1 mL/kg body weight for each dose level. Aliquots of the dosing preparation solutions were stored at ambient conditions and fresh bottles were used for each day of the study. All animals were dosed once daily, by gavage, from days 7 - 19 inclusive of gestation with 1 mL of dosing formulation per kg body weight. The volume given to each animal was adjusted daily according to body weight. Control animals received the appropriate volume of deionised water. All animals receiving the same dose level were dosed sequentially but the order in which each group was dosed was rotated on a daily basis. Dosing was performed in the morning of each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each bulk preparation was analysed prior to the start of dosing to verify the achieved concentration of substance in deionised water. Analysis was by dilution with water followed by HPLC.

Details on mating procedure:

Animals of proven fertility were used for this study. Semen was collected from each male using an artificial vagina. The volume of the sample obtained was measured and then diluted with sufficient physiological saline to inseminate one replicate. One mL of the diluted semen was inseminated into each female using a plastic catheter. Each replicate of females was inseminated with semen from one male. After insemination, each female was given an intravenous injection of 25 IU PROFASI 500 to promote ovulation. Any individual male inseminated a maximum of 16 females (4 replicates). The day of insemination was designated a day 1 of gestation.

Duration of treatment / exposure:

From Day 7 to Day 19 (inclusive) of gestation

Frequency of treatment:

Daily

Duration of test:

Until Day 30 of gestation

Dose / conc.:

1 other: mg test substance cation/kg bw/day

Remarks:

Low dose. Group 2

Dose / conc.:

3 other: mg test substance cation/kg bw/day

Remarks:

Mid dose. Group 3

Dose / conc.:

10 other: mg test substance cation/kg bw/day

Remarks:

High dose. Group 4

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS
All animals were checked on arrival to ensure that they were physically normal externally and were subsequently checked daily.

BODY WEIGHT
The body weight of each animal was recorded on Days 1 and 4, 7 - 19 (inclusive) and on Days 22, 26 and 30 of gestation.

FOOD CONSUMPTION:
The amount of food consumed by each animal was measured by giving a weighed quantity of food on Days 1, 4, 7, 10, 13, 16, 19, 22 and 26 and calculating the amount consumed from the amount left on Days 4, 7, 10, 13, 16, 19, 22, 26 and 30 respectively.

POST-MORTEM EXAMINATIONS:
- Animals which aborted or were considered to be in extremis were killed by an intravenous injection of pentobarbitone sodium solution and given an examination post mortem. Pregnancy status was recorded.
- On Day 30 of gestation the remaining animals were killed by an intravenous injection of pentobarbitone sodium solution. An examination post mortem was performed on all animals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed. The ovaries and uterus were then examined and the following data were recorded:
(1) Number of corpora lutea in each ovary.
(2) Number and position of implantations subdivided into: live foetuses, early intra-uterine deaths and late intra-uterine deaths.

Intra-uterine deaths were classified as early or late intra-uterine deaths.
The implantations were assigned letters of the alphabet to identify their position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.

Fetal examinations:

External examinations: Yes: Each foetus was examined for external abnormalities and cleft palate.
Soft tissue examinations: Yes: All foetuses were then examined internally for visceral abnormalities, sexed, eviscerated and fixed in methanol. After approximately 24 hours the head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities
Skeletal examinations: Yes: The carcasses were then returned to methanol for subsequent processing and staining with Alizarin Red S. The stained foetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed and the result converted to a five point scale. Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external visceral or skeletal examination) defects. Variations were also recorded and classified as minor defects or variants depending on the frequency of occurrence in relation to background data
Head examinations: Yes.

Statistics:

Data relating to animals which were killed in extremis, were non-pregnant or aborted were excluded from the statistical analysis. The following data were considered by analysis of variance:
- Maternal body weight gain
- Maternal food consumption
- The numbers of implantations and live foetuses per female.
- Percentage pre-implantation loss and percentage post-implantation loss

The percentage pre-implantation loss and post-implantation loss were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The analyses of variance were weighted by the denominator in the proportion.

The percentage of implantations which were early intra-uterine deaths and the percentage of implantations which were late intra-uterine deaths (both calculated on an individual litter basis). The percentages were transformed before analysis using the double arcsine transformation and the analysis of variance was weighted by the number of implantations in each litter.
Gravid uterus weight, litter weight and mean foetal weight (calculated on an individual litter basis). The analysis of mean foetal weight was weighted by the number of foetuses in each litter.
Mean manus and pes score per foetus (calculated on an individual litter basis). The analyses were weighted by the number of foetuses examined in each litter. The analyses were weighted by the number of foetuses in each litter.
The percentage of foetuses with minor external/visceral defects only, and minor skeletal defects only (calculated on an individual litter basis). The percentages were transformed before analysis using the double arcsine transformation and the analyses were weighted by the number of foetuses examined in each litter.
The analyses of variance were carried out using the GLM procedure in SAS (1985). Individual treatment group means were compared with the control group mean using Student's t-test based on the error mean square in the analysis.

Indices:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100
Post-implantation loss (%) = (Number of implantations - Number of live foetuses / Number of implantations) x 100

Historical control data:

In addition to historical control data presented in the original report, a supplement to the report contained further information on the variant and minor defect historical control data, together with historical control data for each of the major defects seen in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related changes in the 1 or 3 mg/kg bw/day groups. In the 10 mg/kg bw/day group there were increased numbers of animals with diarrhoea/signs of diarrhoea (although no increase in the numbers of observations), a small increase in the numbers of animals recorded as subdued, a marked increase in the incidence of thinness and a number of observations of mucus, blood and little or no faeces on the tray papers

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Six animals were killed before their scheduled termination dates. One animal from the 1 mg/kg bw/day group was killed on Day 27 following an abortion. Two 10 mg/kg bw/day animals were killed following abortion on Day 20 or Day 29. Three further animals from the 10 mg/kg bw/day group were killed in extremis on Day 17, 21 or 22.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Two 10 mg/kg bw/day females lost weight throughout dosing and then rapidly gained weight during the post-dosing period. Analyses were done both with and without these two females.
There was a clear weight loss in both the 3 mg and 10 mg/kg bw/day dose groups during the first three days of dosing. Subsequent to Day 10, weight loss (or reduced weight gain) was apparent only in certain females in the 10 mg/kg bw/day dose group (most noticeable in the same two animals).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a clear, consistent and dose-related reduction in food consumption in both the 3 mg and 10 mg/kg bw/day dose groups during the first three days of dosing. For the remainder of the dosing period, mean food consumption was lower in the 10 mg/kg bw/day dose group but this was only apparent in certain females. In the 3 mg//kg bw/day dose group, certain females ate more food than others in the group during the latter half of the dosing period.
Post-dosing, some females in both the 3 mg and 10 mg/kg bw/day dose groups ate more food with consequently higher mean food consumption. The increases in food consumption during the latter period (3 mg and 10 mg/kg bw/day groups) may indicate a compensatory effect for the earlier reductions.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The major findings were changes in the alimentary tract which were seen most commonly in the intercurrent deaths in the 10 mg/kg bw/day group. Of these, the most important change was haemorrhage and/or ulceration in the stomach which was also seen in two animals (one each in the 1 and 10 mg/kg bw/day groups) surviving to termination. Prominent reticular pattern in the liver was also seen in a number of animals, particularly in the intercurrent deaths. One unusual finding in the intercurrent deaths was changes in the intestinal blood vessels; in one animal, the vessels in the caecum appeared to have leaked.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Three animals aborted, one in the 1 and two in the 10 mg/kg bw/day groups respectively.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: test substance cation species

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Original value presented in study

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: pure test substance

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Reduction in number of live offspring:

effects observed, non-treatment-related

Changes in sex ratio:

effects observed, non-treatment-related

Changes in litter size and weights:

effects observed, non-treatment-related

Changes in postnatal survival:

effects observed, non-treatment-related

External malformations:

no effects observed

Description (incidence and severity):

The overall incidence of external defects was unaffected by treatment with test substance.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Overall, there was a dose-related increase in the percentage of foetuses with minor skeletal defects. The difference between the control group and the 3 mg and 10 mg/kg bw/day dose groups attained statistical significance using a foetus-based analysis (Fisher's Exact Test). However, the difference between the control group and the 1 mg/kg bw/day dose group was marginal and not statistically significant. Using a litter-based analysis (double arcsine transformation and analysis of variance) which is considered to be more appropriate for defect data, there were no statistically significant differences compared with the control group for any of the test groups.
Specific minor defects to show increased statistical incidence in any dose group were:
- Not ossified 6th sternebra, and
- partially ossified 6th sternebra.
Both these defects were statistically significantly increased at the 10 mg/kg bw/day dose level. Partially ossified 6th sternebra was also higher than control at the 3 mg substance /kg/day dose level. A re-examination of the ventral tubercle was carried out because the incidence reported was found to be inconsistent with historical data, i.e. no partially ossified ventral tubercles from control foetuses have been seen either before or after this study. It was the opinion of the re-evaluator that all of the foetuses examined exhibited ventral tubercles which were within normal limits and all are considered to be fully ossified. Therefore no ventral tubercles in this study are considered to be partially ossified following this re-evaluation.
Not ossified 6th sternebra was statistically significantly increased at the 1 mg/kg bw/day dose level but the frequency was lower at 3 mg than 1 mg/kg bw/day and hence there was no dose response at least at the two lower levels. These two defects in combination showed an apparent dose response, but all values were within historical control values. The control value for the present study appears to be unusually low. Only three foetuses did not have a skeletal variant. Only three foetuses did not have a skeletal variant.
The incidence of foetuses with major defects was 2, 8, 2 and 4 in each dose group respectively. Since most specific major defects occurred as single incidences in one (or two) treatment groups they were considered to be spontaneous occurrences and not as a result of treatment with substance.
There was a clear increase in the proportion of foetuses with pes score 2 in the 10 mg/kg bw/day dose group. There was a similar increase in the proportion of foetuses with manus score 2, also in the 10 mg substance /kg/day dose group. This did not achieve statistical significance although was reflected in the mean score. There was no evidence of an effect at the two lower doses.
There was a statistically significant increase in the incidence of 27 pre-sacral vertebrae in the 10 mg/kg bw/day dose group although this was at the upper end of the control range. There was an increased incidence of 13th extra ribs in both the 3 mg and 10 mg/kg bw/day dose group. Extra 13th ribs showed a similar increase in incidence to that of 27 pre-sacral vertebrae and again the incidence at 10 mg/kg bw/day was at the upper end of the control range.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The overall incidence of external/visceral defects was unaffected by treatment with test substance. Three specific minor external/visceral defects observed were:
- Friable liver which was increased in the 10 mg/kg bw/day group.
- Mottled liver which was increased in the 10 mg/kg bw/day group and tended to occur in the foetuses with friable liver.
- Gall bladder: small blood clots attached - the incidence of foetuses with this defect in the 3 mg/kg bw/day dose group was well above the historical control range but there was clearly no dose response.

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: sternebra ossification

Remarks on result:

other:

Remarks:

Original value presented in study

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: sternebra ossification

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

Diet/dosage preparation and analysis

- Concentration analysis: Mean test substance concentrations were within 7 % of the nominal values.

- Homogeneity: The preparations formed solutions in deionised water.

- Stability: The chemical stability of test substance in deionised water after an interval of 23 days (which is the same as the dosing period in the present study) was assessed in a preliminary study in the rabbit and found to be satisfactory.

Calculation of key result

The original effect levels were expressed as cation species of the test substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

The NOAEL for maternal toxicity = 1.868 x 1 mg/kg bw/day = 1.9 mg/kg bw/day

The NOAEL for fetal developmental toxicity = 1.868 x 3 mg/kg bw/day = 5.6 mg/kg bw/day

Table 1: Intergroup comparison of body weight gain (g) all females

	Dose Level of test substance (mg/kg/day)
Period (days)	0 (control)	1	3	10 (all females)	10 mg/kg/day Excluding 2 females
Initial wt (1)	3418.1	3401.3	3344.5	3368.2	3357.1
Pre-dosing (1 – 7)	130.2	131.0	158.4	171.9	165.0
During dosing (7 – 19)	237.4	309.9	236.4	47.5**	175.1
Post dosing (19 – 30)	312.4	387.1	293.3	424.8*	352.4
Overall (1 – 30)	680.1	828.0*	688.2	644.3	692.5

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 2: Intergroup comparison of food consumption (g/day)

	Dose Level of test substance (mg/kg/day)
Period (days)	0 (control)	1	3	10
Pre-dosing (1 – 7)	173.2	162.4	168.8	172.3
During dosing (7 – 19)	163.4	158.1	150.1	96.0**
Post dosing (16 – 30)	164.1	170.4	181.0*	186.6*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 3: Caesarean section observations for all pregnant females

Observation	Dose Level of test substance (mg/kg/day)
Observation	0 (control)	1	3	10
Animals inseminated	20	20	20	20
Animals Pregnant	18	16	20	17
Number of females that aborted	0	1	0	2
Number of intercurrent deaths (including females with abortions)	0	0	0	3^{^}
Number of females with live foetusesin uteroat termination	18	15	20	13
Corpora Lutea/Dam	11.50	11.93	9.35	10.46
Pre-implantation Loss (%)	16.4	12.3	18.2	14.7
Number of females affected	11/18	9/15	12/20	5/13
Mean number of implantations	9.61	10.47	7.65*	8.92
Post-implantation Loss (%)	15.0	14.6	15.7	17.2
Number of females affected	13/18	9/15	14/20	8/13
Mean number of live foetuses	8.17	8.93	6.45*	7.38
Number of intra-uterine deaths % Early Late	6.9 8.1	3.8 10.8	6.5 9.2	9.5 7.8
Intra-uterine deaths/Dam
Early (Proportion of litters affected)	8/18	4/15	7/20	5/13
Late (Proportion of litters affected)	6/18	7/15	8/20	5/13
Total number of live foetuses	147	134	129	96
Sex Ratio (% Males per litter)	54.4	49.6	49.6	47.9
Mean gravid uterus weight (g)	483.1	539.7	394.9*	413.3
Mean Litter Weight (g)	325.1	344.8	271.4	273.6
Mean Foetal Weight (g)	39.81	38.59	42.08	37.05

^ Including one animal that was not pregnant.

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 4 Intergroup comparison of selected foetal defects and variants

Observation	Dose Level of test substance (mg/kg/day)
Observation	0 (control)	1	3	10
No. of litters examined	18	15	20	13
No. of foetuses examined	147	134	129	96
Total incidence of major defects	2	8	2	4
No. of foetuses showing major external/visceral defects	2	8*	2	3
No. of foetuses showing major skeletal defects	1	3	1	1
No. of foetuses showing minor external/visceral defects only	60	56	64	39
No. of foetuses showing minor skeletal defects only	64	65	71*	53*
Liver – friable (foetus/litter)	8/5	7/2	5/2	14*/3
Gall bladder – small blood clot(s) attached (foetus/litter)	18/8	21/9	33**/13	14/8
Vertebral column – 27 pre-sacral vertebrae (foetus/litter)	46/14	54/12	49/15	49**/12
Sternebrae – not ossified 6^th(foetus/litter)	0/0	6*/2	3/3	6/5
Sternebrae – partially ossified 6^th(foetus/litter)	9/6	14/8	19*/8	16**/8
Extra ribs – 13^thnormal length (foetus/litter)	76/15	61/13	89**/18	71**/12
Mean manus score	1.61	1.60	1.62	1.80
Mean pes score	1.43	1.45	1.40	1.65*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Conclusions:

Test substance administered to rabbits by gavage during gestation at a concentration of 10 mg test substance ion/kg bw/day, equivalent to 19 mg pure test substance/kg bw/day, caused maternal toxicity and foetotoxicity. Mild maternal toxicity was seen with 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day. The NOAEL for maternal toxicity was set at 1 mg test substance ion/kg bw/day, corresponding to 1.9 mg pure test substance/kg bw/day. A dose of 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day, was considered to be a no-effect level for embryonic and foetal development.

Executive summary:

A study, complying to GLP and OECD guideline 414, investigated the effect of the test substance when administered by gavage during the period of organogenesis on the embryonic and foetal development of the rabbit. Groups of 20 young adult female New Zealand White rabbits were dosed by gavage with 1, 3 or 10 mg test substance cation/kg bw/day in deionised water from Days 7 - 19 (inclusive) of gestation which thus included the period of major organogenesis. A control group of animals received deionised water alone. The day of insemination was designated Day 1 of gestation. On Day 30 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, killed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

Administration of 10 mg/kg bw/day resulted in maternal toxicity which was apparent as weight loss and reduced food consumption. Five animals (including two which aborted) from this group were killed prematurely. Similar but less severe effects on body weight and food consumption were seen at 3 mg/kg bw/day. There were no treatment-related effects on numbers of corpora lutea, pre-or post-implantation loss, litter size, sex distribution, gravid uterus weight or litter weight. Foetal weight was marginally lower at 10 mg/kg bw/day. Sixteen foetuses were seen with major abnormalities (2, 8, 2 and 4 foetuses in the control, 1, 3 and 10 mg/kg bw/day groups, respectively). There was no evidence that the type or distribution of these was related to treatment. The overall incidence of foetuses with minor external/visceral defects was not affected by treatment although the incidence of mottled and friable livers was increased at 10 mg/kg bw/day. There was a small increase in the percentage of foetuses with minor skeletal defects at 3 and 10 mg test substance/kg/day. The incidences were not statistically significant using a litter-based method of analysis. Specifically, there were increased numbers of partially ossified 6th sternebra at both levels while not ossified 6th sternebra showed an increase at 10 mg/kg bw/day only. A re-examination of the ventral tubercle was carried out because the incidence reported was found to be inconsistent with historical data, i.e. no partially ossified ventral tubercles from control foetuses have been seen either before or after this study. It was the opinion of the re-evaluator that all of the foetuses examined exhibited ventral tubercles which were within normal limits and all are considered to be fully ossified. Therefore no ventral tubercles in this study are considered to be partially ossified following this re-evaluation. The frequency of the skeletal variant 27 pre-sacral vertebrae was increased at 10 mg/kg bw/day while the incidence of extra thoracic ribs was increased at both 3 and 10 mg/kg bw/day although in all cases these were within recent historical control incidences. Manus and pes scores were increased at 10 mg test substance cation/kg/day. These scores, which are generally sensitive indicators of reduced ossification, were similar in the 1 and 3 mg/kg bw/day groups to those of the controls.

Test substance administered to rabbits by gavage during gestation at 10 mg test substance cation/kg bw/day, corresponding to 19 mg pure test substance/kg bw/day, caused maternal toxicity and foetotoxicity. Mild maternal toxicity was seen at 3 mg test substance cation/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day. The NOAEL for maternal toxicity was determined to be 1.9 mg pure test substance /kg bw/day. The dose of 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance salt/kg bw/day, was considered to be the NOAEL for embryonic and foetal development.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb 1988 to 29 Mar 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2004

GLP compliance:

yes

Limit test:

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: 212 – 326 g
- Housing: Individually housed in stainless steel suspended cages.
- Diet: Ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: Not applicable

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24
- Humidity (%): 30 - 54
- Air changes (per hr): minimum of 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 22 Feb 1988 To: 29 Mar 1988

Route of administration:

oral: gavage

Vehicle:

other: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Test substance was formulated in deionised water. The concentration of test substance was adjusted to give a constant volume of 1 mL/100 g body weight for each dose level. An appropriate volume of deionised water was added to a weighed amount of substance liquor to provide one preparation per dose level and stored in ambient conditions. Fresh bottles were used for each day of the study.

DOSAGE ADMINISTRATION
All animals were gavage dosed from Days 7 - 16 (inclusive) of gestation with 1 mL of dosing suspension per 100 g body weight. The volume given to each animal was adjusted daily according to body weight. Control animals received the appropriate volume of deionised water. All animals receiving the same dose level were dosed sequentially and the groups were dosed in numerical order. Dosing was performed in the morning of each day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY OF TEST SUBSTANCE FORMULATIONS
A sample of each preparation was analysed prior to the start of dosing to verify the achieved concentrations of subtance in deionised water. The chemical stability of test substance in deionised water was determined by re-analysis of the dosing formulation of each dose level (nominally 0.4, 1.2 and 4.0 mg/mL) after an interval of 29 days. Analysis was performed by dilution with distilled water followed by HPLC.

Details on mating procedure:

Each female rat was paired overnight at the Breeding Unit with an unrelated male of the same strain. On the following morning, vaginal smears from these females were examined for the presence of sperm. The day when spermatozoa were detected was designated Day 1 of gestation and on this same day successfully mated females were delivered to the experimental unit at CTL. A total of 96 mated females was supplied over a two week period. Eight female rats were supplied on each of twelve days.

Duration of treatment / exposure:

From Day 7 to Day 16 of gestation

Frequency of treatment:

Daily

Duration of test:

Until Day 22 of gestation

Dose / conc.:

4 other: mg test substance cation/kg bw /day

Remarks:

Low dose. Group 2

Dose / conc.:

12 other: mg test substance cation/kg bw/day

Remarks:

Mid dose. Group 3

Dose / conc.:

40 other: mg test substance cation/kg bw/day

Remarks:

High dose. Group 4

No. of animals per sex per dose:

Control animals:

yes, concurrent vehicle

Details on study design:

ANIMAL ASSIGNMENT
The study was divided into 24 replicates (randomised blocks) with each replicate containing one rat from each dosage group. Cages within the replicates were assigned to one of the four groups using computer-generated random number permutations. The individual animal numbers were then assigned sequentially within the relevant groups. On arrival (Day 1 of gestation), each rat was allocated to a cage (and therefore a treatment group) randomly within the replicate and individually identified by ear punching with the number assigned to it from the experimental design. Replicates were filled sequentially with two replicates added to the study on each of the twelve days on which rats were received.

Maternal examinations:

CAGE SIDE OBSERVATIONS
All animals were checked on arrival to ensure that they were physically normal externally and were subsequently observed daily. Any changes in behaviour or clinical condition were recorded daily during the dosing period and on those days when they were weighed.

BODY WEIGHTS
The body weight of each animal was recorded on Days 1 and 4 and subsequently on Days 7 - 16 (inclusive) and on Days 19 and 22 of gestation.

FOOD CONSUMPTION
The amount of food consumed by each animal over three day periods was measured by giving a weighed quantity of food contained in a glass jar on Days 1, 4, 7, 10, 13, 16 and 19 and calculating the amount consumed from the residue on Days 4, 7, 10, 13, 16, 19 and 22 respectively.

POST-MORTEM EXAMINATIONS:
- On Day 22 of gestation, all the remaining animals were killed by over-exposure to halothane BP (FLUOTHANE). An examination post mortem was performed on all animals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed. The ovaries and uterus were then examined and the following data recorded:
(1) Number of corpora lutea in each ovary.
(2) Number and position of implantations subdivided into: live foetuses, early intra-uterine deaths and late intra-uterine deaths.

Intra-uterine deaths were classified as follows: early intra-uterine deaths showed decidual or placental tissue only. Late intra-uterine deaths showed embryonic or foetal tissue in addition to placental tissue. The implantations were assigned letters of the alphabet to identify their position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.

Fetal examinations:

Each foetus was weighed and individually identified within the litter by means of a cardboard tag. After weighing, the foetuses were killed with an intra-cardiac injection of pentobarbitone sodium solution, 200 mg/mL (EUTHATAL).
External examinations: Yes: Each foetus was examined for external abnormalities and for cleft palate. All foetuses were then examined internally for visceral abnormalities by micro-dissection under magnification, sexed, eviscerated and fixed in methanol.
Soft tissue examinations: Yes: After approximately 24 hours fixation, the head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities. The carcasses were then returned to methanol for subsequent processing and staining with Alizarin Red S.
Skeletal examinations: Yes: The stained foetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed according to a four point scale.
Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external, visceral or skeletal examination) defects. Other common variations (depending on the historical frequency of occurrence in rats of this strain) were classified as variants.

Statistics:

Data relating to animals which littered, died or were non-pregnant were excluded from the statistical analysis. The following data were considered by analysis of variance:
- Maternal body weight gain
- Maternal food consumption
- The numbers of implantations and live foetuses per female.

The percentage pre-implantation loss and post-implantation loss were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The analyses of variance were weighted by the denominator in the proportion.

The percentage of implantations which were early intra-uterine deaths (calculated on an individual litter basis). The percentage was transformed before analysis using the double arcsine transformation and the analysis of variance was weighted by the number of implantations in each litter.
Gravid uterus weight, litter weight and mean foetal weight (calculated on an individual litter basis). The analysis of mean foetal weight was weighted by the number of foetuses in each litter.
Mean manus and pes score per foetus (calculated on an individual litter basis). The analyses were weighted by the number of foetuses examined in each litter.
The percentage of foetuses with minor external/visceral defects only, external/visceral variants and minor skeletal defects only (calculated on an individual litter basis). The percentages were transformed before analysis using the double arcsine transformation and the analyses were weighted by the number of foetuses examined in each litter.

The analyses of variance allowed for the replicate structure of the study design and were carried out using the GLM procedure in SAS (1985). Unbiased estimates of the treatment group means were provided by the least square means (LSMEANS option in SAS). Individual treatment group means were compared with the control group mean using Student's t-test based on the error mean square in the analysis.

Indices:

Historical control data:

Data from other studies conducted during 1986 – 1988 in the same laboratory with the same strain of rats were used to compare the incidence of selected findings in the current study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 40 mg/kg bw/day group, the majority of animals showed piloerection during the second half of the study. Three animals in this group also appeared subdued during the same period although the incidence was low and could not conclusively be attributed to the test substance. The animal found dead on Day 15 appeared thin and hunched with piloerection and urinary incontinence on the previous day. Other observations were of low incidence or not dose-related and of the type commonly seen in this strain of rat.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal in the 40 mg/kg bw/day group was found dead on Day 15. All other animals survived for the duration of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A dose-related, statistically significant decrease in body weight gain compared to control values was seen across all dose groups during the first three days of dosing. There was a clear statistically significant decrease in body weight gain in the 40 mg/kg bw/day dose group throughout the dosing period, with the majority of the animals losing weight initially. Evidence of recovery was seen in this group during the post-dosing period but the overall weight gain remained statistically significantly lower than controls. The effect seen at 4 and 12 mg/kg bw/day was evident only during the first few days of dosing and was very mild at 4 mg/kg bw/day

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A dose-related, statistically significant reduction in food consumption compared to controls was seen across all dose groups during the first three days of dosing. This effect persisted throughout the dosing period for animals in the 12 and 40 mg/kg bw/day groups and continued in the top dose for the first three days post-dosing. It did not persist in the 4 mg/kg bw/day group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were seen.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Mean foetal weight and litter weight were statistically significantly reduced compared to controls at the 40 mg/kg bw/day dose level. The gravid uterus weight at this level was also lower as a result of the reduced litter weight. No notable differences between test and control groups were seen at either the 12 or 4 mg/kg bw/day level.

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: test substance cation species

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Original value presented in study

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: pure test substance

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration of 40 mg test substance cation species/kg bw/day produced significant reductions in foetal weight.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Administration of 40 mg/kg bw/day produced significant reductions in litter weight.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The incidence of external defects was unaffected by treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Major defects were seen in 8 foetuses, the incidence being 1, 2, 1 and 4 in the 0, 4, 12 and 40 mg/kg bw/day groups, respectively. There was no evidence that the type or distribution of these abnormalities was related to the treatment.
Overall, there was a clear increase in foetuses with minor skeletal defects in the 40 mg/kg bw/day group. Skeletal variants and manus and pes scores were also increased at this dose level. These findings indicate poorer ossification at this top dose level. Evidence of reduced ossification was also seen across the lower dose groups in the dose-related differences seen in the incidence of calcaneum not ossified, not ossified odontoid and partially ossified transverse processes of the 4th lumbar vertebrae. None of these variants achieved statistical significance at the low dose level. Although the findings at 4 and 12 mg/kg bw/day were consistent with the lower end of a dose response relationship, the differences seen were within recent background control incidences and/or considered too small to be of biological significance.

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of minor external and visceral defects was unaffected by treatment with test substance at the 4 and 12 mg/kg bw/day dose levels. The possibility that the higher incidence of foetuses with haemorrhagic kidney seen at the 40 mg/kg bw/day dose level was due to the administration of test substance is unlikely.

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

other: test substance cation species

Sex:

male/female

Basis for effect level:

other: skeletal defects

Remarks on result:

other:

Remarks:

Original value presented in study

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: skeletal defects

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: hindlimb

other: skeletal defects

Description (incidence and severity):

Poor ossification

Developmental effects observed:

Diet/dosage preparation and analysis

- Concentration analysis: The mean achieved concentrations of the substance were found to be within ±7.5 % of nominal values.

- Homogeneity: The preparations formed solutions in deionised water.

- Stability: The chemical stability of subtance in deionised water for each dosing formulation was found to be satisfactory over a 29 day analysis interval which is greater than the period of 25 days over which the formulations were used in the study.

Table 1: Intergroup comparison of body weight gain (g)

	Dose Level of test substance cation species [mg/kg/day]
Period (days)	0 (control)	4	12	40
Initial wt (1)	255.0	259.8	258.6	258.3
Pre-dosing (1 – 7)	28.0	29.5	26.3	31.1
During dosing (7 – 16)	49.1	45.0	43.5*	7.9**
Post dosing (16 – 22)	53.0	56.6	53.9	62.3
Overall (1 – 22)	130.1	131.2	123.7	101.5**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 2: Intergroup# comparison of food consumption (g/day)

	Dose Level of test substance cation species [mg/kg/day]
Period (days)	0 (control)	4	12	40
Pre-dosing (1 – 7)	22.7	23.0	22.5	23.7
During dosing (7 – 16)	28.2	27.4	26.0**	17.6**
Post dosing (16 – 22)	28.3	29.0	28.5	26.5

# Values from one group 2 female excluded as this animal consistently ate more than other females

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 3: Caesarean section observations for all pregnant females

Observation	Dose Level of test substance cation species [mg/kg/day]
Observation	0 (control)	4	12	40
Animals assigned (mated)	24	24	23	24
Animals pregnant	24	24	23	20
Corpora lutea/dam	14.0	13.4	13.6	13.6
Pre-implantation loss (%)	7.8	5.3	9.0	8.4
Number of females affected	10/24	11/24	13/23	10/20
Mean number of implantations	12.9	12.7	12.3	12.5
Post-implantation loss (%)	3.9	4.3	5.3	4.4
Number of females affected	8/24	9/24	11/23	7/20
Mean number of live foetuses	12.4	12.1	11.6	12.0
% Intra-uterine deaths/dam
Early (proportion of litters affected)	3.6 (8/24)	3.3 (7/24)	4.2 (9/23)	3.2 (4/20)
Late (proportion of litters affected)	0.3 (1/24)	1.0 (3/24)	1.1 (3/23)	1.2 (3/20)
Total number of live foetuses	297	291	269	239
Sex ratio (% males per litter)	51.9 (154/297)	51.2 (149/291)	52.4 (141/269)	51.0 (122/239)
Mean gravid uterus weight (g)	84.7	85.5	81.6	77.0
Mean litter weight (g)	59.6	59.4	56.1	52.6*
Mean foetal weight (g)	4.83	4.90	4.85	4.41**

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Table 4: Intergroup comparison of selected foetal defects and variants

Observation	Dose Level of test substance cation species [mg/kg/day]
Observation	0 (control)	4	12	40
No. of litters examined	24	24	23	20
No. of foetuses examined	297	291	269	239
Total incidence of major defects	1	2	1	4
No. of foetuses showing major skeletal defects	1	1	1	1
No. of foetuses showing minor skeletal defects only	106	102	97	132**
Incidence of haemorrhagic kidneys (foetus/litter)	0	1 (1)	0	5* (4*)
Incidence of calcaneum not ossified (foetus/litter)	133 (22)	139 (21)	157** (22)	149** (19)
Incidence of odontoid not ossified (foetus/litter)	107 (22)	114 (19)	110 (17)	102 (19)
Incidence of partially ossified 4^thlumbar transverse process (foetus/litter)	232 (24)	210 (24)	182 (22)	128 (19)
Mean manus score	2.20	2.25	2.18	2.52**
Mean pes score	2.92	2.93	2.92	2.98

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Calculation of key result

The NOAEL for maternal toxicity = 1.868 x 4 mg/kg bw/day= 7.47 mg/kg bw/day

The NOAEL for developmental toxicity = 1.868 x 12 mg/kg bw/day = 22.4 mg/kg bw/day

Conclusions:

There was no evidence that the test substance is teratogenic to the rat at any of the dose levels tested in this study. Test substance administered to rats by gavage throughout gestation at 40 mg test substance cation/kg bw/day caused maternal toxicity and foetotoxicity. Mild transient maternal toxicity was seen at both 12 and 4 mg/kg bw/day. The NOAEL for maternal toxicity was set at 4 mg/kg bw/day. The NOAEL for developmental toxicity was set at 12 mg test substance cation/kg bw/day, equivalent to 22.4 mg pure test substance/kg bw/day registered substance.

Executive summary:

A study, complying to GLP and OECD guideline 414, investigated the effect of the test substance when administered by gavage during the period of organogenesis on the embryonic and foetal development of the rat. Groups of 24 young adult, Wistar-derived (Alpk:APfSD) female rats were dosed by gavage with concentrations of 4, 12 or 40 mg test substance cation/kg bw/day in deionised water from Days 7 - 16 (inclusive) of gestation. The control group received deionised water alone. The day of confirmation of mating was designated Day 1 of gestation. On Day 22 of gestation, the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, killed, sexed, eviscerated, examined for external and visceral abnormalities, and stained for skeletal examination.

Administration of 40 mg/kg bw/day resulted in maternal toxicity, manifest as a reduction in weight gain and food consumption during the dosing period. There was an initial but transient effect on weight gain and food consumption at 4 and 12 mg/kg bw/day dose levels during the dosing period. Administration of 40 mg/kg bw/day produced significant reductions in foetal weight, litter weight and gravid uterus weight. A higher incidence of foetuses with unilateral haemorrhagic kidney seen at this dose level was unlikely to be compound-related. Overall, there was a clear increase in foetuses with minor skeletal defects, skeletal variants and manus and pes scores at 40 mg/kg bw/day, indicative of poorer ossification. Evidence of reduced ossification was also seen in the lower dose groups in the incidence of not ossified calcaneum, not ossified odontoid and partially ossified transverse processes of the 4th lumbar vertebrae. However, these changes in ossification at 4 and 12 mg/kg bw/day were considered too small to be of biological significance.

In conclusion, there was no evidence that the test substance is teratogenic to the rat at any of the dose levels tested in this study. Test substance administered to rats by gavage throughout gestation at a concentration of 40 mg test substance cation/kg bw/day caused maternal toxicity and foetotoxicity. Mild transient maternal toxicity was seen at both 12 and 4 mg/kg bw/day. The NOAEL for maternal toxicity was set at 4 mg/kg bw/day. The NOAEL for developmental toxicity was set at 12 mg test substance cation/kg bw/day, equivalent to 22.4 mg pure test substance/kg bw/day registered substance.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 5.64 mg/kg bw/day
Study duration:: subchronic
Species:: rabbit
Quality of whole database:: GLP compliant OECD 414 study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Toxicity to reproduction: other studies

Additional information

Developmental toxicity study - OECD 414, rabbit (Hodge 1989)

The study, complying to GLP and OECD guideline 414, investigated the effect of the test substance when administered by gavage during the period of organogenesis on the embryonic and foetal development of the rabbit. Groups of 20 young adult female New Zealand White rabbits were dosed by gavage with 1, 3 or 10 mg test substance cation/kg bw/day in deionised water from Days 7 - 19 (inclusive) of gestation which included the period of major organogenesis. A control group of animals received deionised water alone. The day of insemination was designated Day 1 of gestation. On Day 30 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, killed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

Administration of 10 mg/kg bw/day resulted in maternal toxicity which was apparent as weight loss and reduced food consumption. Five animals (including two which aborted) from this group were killed prematurely. Similar but less severe effects on body weight and food consumption were seen at 3 mg/kg bw/day. There were no treatment-related effects on numbers of corpora lutea, pre-or post-implantation loss, litter size, sex distribution, gravid uterus weight or litter weight. Foetal weight was marginally lower at 10 mg/kg bw/day. Sixteen foetuses were seen with major abnormalities (2, 8, 2 and 4 foetuses in the control, 1, 3 and 10 mg/kg bw/day groups respectively). There was no evidence that the type or distribution of these was related to treatment. The overall incidence of foetuses with minor external/visceral defects was not affected by treatment although the incidence of mottled and friable livers was increased at 10 mg/kg bw/day. There was a small increase in the percentage of foetuses with minor skeletal defects at 3 and 10 mg test substance cation/kg/day. The incidences were not statistically significant using a litter-based method of analysis. Specifically, there were increased numbers of partially ossified 6th sternebra at both levels while not ossified 6th sternebra showed an increase at 10 mg/kg bw/day only. A re-examination of the ventral tubercle was carried out because the incidence reported was found to be inconsistent with historical data, i.e. no partially ossified ventral tubercles from control foetuses have been seen either before or after this study. It was the opinion of the re-evaluator that all of the foetuses examined exhibited ventral tubercles which were within normal limits and all are considered to be fully ossified. Therefore, no ventral tubercles in this study are considered to be partially ossified following this re-evaluation. The frequency of the skeletal variant 27 pre-sacral vertebrae was increased at 10 mg/kg bw/day while the incidence of extra thoracic ribs was increased at both 3 and 10 mg/kg bw/day although in all cases these were within recent historical control incidences. Manus and pes scores were increased at 10 mg test substance cation/kg/day. These scores, which are generally sensitive indicators of reduced ossification, were similar in the 1 and 3 mg/kg bw/day groups to those of the controls.

Test substance administered to rabbits by gavage during gestation at a concentration of 10 mg test substance cation/kg bw/day, equivalent to 19 mg pure test substance/kg bw/day caused maternal toxicity and foetotoxicity. Mild maternal toxicity was seen with 3 mg test substance ion/kg bw/day equivalent to 5.64 mg pure test substance/kg bw/day. The NOAEL for maternal toxicity was 1.9 mg pure test substance/kg bw/day. A dose of 3 mg test substance cation/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day, was considered to be the NOAEL for embryonic and foetal development.

Developmental toxicity study - OECD 414, rat (Wickramarathne 1989)

This study, complying to GLP and OECD guideline 414, investigated the effect of the test substance when administered by gavage during the period of organogenesis on the embryonic and foetal development of the rat. Groups of 24 young adult, Wistar-derived (Alpk:APfSD) female rats were dosed by gavage with 4, 12 or 40 mg test substance cation/kg bw/day in deionised water from Days 7 to 16 (inclusive) of gestation. The control group received deionised water alone. The day of confirmation of mating was designated Day 1 of gestation. On Day 22 of gestation, the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, killed, sexed, eviscerated, examined for external and visceral abnormalities, and stained for skeletal examination.

In conclusion, there was no evidence that the test substance is teratogenic to the rat at any of the dose levels tested in this study. Test substance administered to rats by gavage throughout gestation at 40 mg/kg bw/day caused maternal toxicity and foetotoxicity. Mild transient maternal toxicity was seen at both 12 and 4 mg/kg bw/day. The NOAEL for maternal toxicity was set at 4 mg test substance cation/kg bw/day, equivalent to 7.47 mg/kg bw/day pure test substance. The NOAEL for developmental toxicity was set at 12 mg test substance cation/kg bw/day, equivalent to 22.4 mg/kg bw/day pure test substance.

Justification for classification or non-classification

Based on the available information, classification for reproductive toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Additional information

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

	Dietary Concentration of the test substance ion (ppm)
	Males				Females
Week	0	16	80	400/240	0	16	80	400/240
F0 parent:initial weight	83.6	86.0	84.7	82.0	78.4	80.8	77.1	80.1
4	208.8	211.6	208.7	165.2**	115.0	111.8	113.5	98.1**
8	328.9	331.8	330.2	270.3**	162.2	160.8	162.4	143.9**
Final weight	476.9	485.8	480.3	416.2**	263.8	266.5	264.9	247.4**
F1 parent:initial weight	122.8	123.1	119.3	74.1	111.4	110.8	104.4	73.4
4	202.3	199.0	199.2	168.3**	99.7	95.0	102.5	106.3
8	304.1	301.7	303.2	285.4*	139.6	137.0	144.4	155.6**
Final weight	473.5	472.7	464.9	413.0**	268.6	263.5	264.8	247.3**
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

	Dietary Concentration of the test substance ion (ppm)
F0 females	0	16	80	400/240
Day 1 (initial weight)	273.9	275.0	271.4	254.4**
Day 22 (final weight)	395.1	399.6	390.4	347.3**
F1 females
Day 1 (initial weight)	276.7	275.9	272.9	257.4**
Day 22 (final weight)	395.8	400.5	397.5	378.5*

	Dietary Concentration of test substance ion (ppm)
F0 females	0	16	80	400/240
Day 1 (initial weight)	304.2	311.3	296.7	269.4**
Day 22 (final weight)	344.6	345.6	340.0	277.6**
F1 females
Day 1 (initial weight)	301.9	305.3	298.9	287.8
Day 22 (final weight)	341.1	342.1	337.4	310.4**

	Dietary Concentration of the test substance ion (ppm)
Generation: day	0	16	80	400/240
F1A litter: day 1	10.8 (29)	10.6 (26)	11.0 (29)	9.2* (26)
36	9.6 (29)	9.5 (26)	9.9 (29)	8.1* (25)
F2A litter: day 1	10.5 (27)	11.1 (27)	10.7 (29)	10.8 (27)
36	8.8 (27)	10.4* (27)	9.2 (29)	9.3 (27)
Number of pups (number of litters)

	Dietary Concentration of the test substance ion (ppm)
Generation: obs	0	16	80	400/240
F1A : Signs of urinary incon. No. obs	1	1	3	15
No. litters (period of days seen)	1 (22)	1 (22)	1 (29)	6 (29 – 39)
F1A : Distended abdomen. No. obs	-	-	-	22
No. litters (period of days seen)				4 (29 – 36)
F1A : Piloerection. No. obs	2	-	-	14
No. litters (period of days seen)	1 (22)			3 (29 – 39)
F2A : Signs of urinary incon. No. obs	-	-	1	8
No. litters (period of days seen)			1 (29)	2 (29 –36)
Obs – observations incon. – incontinence No. – number of

Generation/Sex	Dietary Concentration of the test substance ion (ppm)
	16	80	400/240^#
F0 Parents: Males	1.6	7.9	38.7
Females	1.7	8.4	40.4
F1 Parents: Males	1.5	7.8	34.7
Females	1.7	8.4	35.7

Observation	Dietary Concentration of the test substance ion (ppm)
Observation	0	16	80	400
Males: (proportion of fertile animals) %	93	82	97	87
Females: (proportion of fertile animals) %	100	100	100	100
Mean precoital interval (days)	3.10	2.35	2.52	3.19
Females placed with males	30	28	30	30
Pregnant	30	28	30	30
Median gestation interval (days)	22.2	22.0	22.0	22.1
Number of litters with all pups live born	26	21	25	23
Number of litters with all pups surviving to day 22	13	15	15	12

	Dietary Concentration of the test substance ion (ppm)
	Males				Females
Generation: Day	0	16	80	400	0	16	80	400/240
F1A:initial weight (day 1)	6.1	6.1	5.9	5.9	5.7	5.7	5.6	5.6
5	2.8	3.0	2.7	3.1	2.7	3.0	2.6	2.8
11	12.0	12.0	11.4	11.4	11.4	11.4	11.1	10.8
22	34.8	34.5	32.1	27.6**	32.6	32.6	30.7	26.7**
36	119.4	119.4	111.0*	68.6**	105.4	104.7	98.9	66.0**
F2A:initial weight (day 1)	6.1	6.0	6.0	6.1	5.7	5.7	5.7	5.8
5	3.0	3.1	3.2	3.1	3.0	3.0	3.1	2.9
11	13.6	12.6	13.8	12.5	13.1	12.2	13.5	12.2
22	39.1	35.2*	38.5	34.1**	38.0	34.3*	37.0	33.0**
36	129.9	122.4*	128.7	111.9**	113.4	108.4	111.9	100.4**
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

Diquat dibromide

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Link to relevant study records

Reference

Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Effect on developmental toxicity: via oral route

Effect on developmental toxicity: via inhalation route

Effect on developmental toxicity: via dermal route

Toxicity to reproduction: other studies

Additional information

Justification for classification or non-classification

Additional information

Referenceopen all close all