Diquat dibromide

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Diquat dibromide

EC Number:

201-579-4

EC Name:

Diquat dibromide

Cas Number:

85-00-7

Molecular formula:

C12H12N2.2Br

IUPAC Name:

1,1'-ethylene 2,2'-bipyridyldiylium dibromide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

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.

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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.

2

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.

3

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

4

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.

5

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.

6

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.

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

1

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.

2

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.

3

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

4

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.

5

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.

6

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.

Target system / organ toxicity (P1)

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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.

2

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.

Target system / organ toxicity (F1)

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

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

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.

2

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

Target system / organ toxicity (F2)

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

Overall reproductive toxicity

1

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

2

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

Any other information on results incl. tables

Calculation of key result

The original effect levels were expressed as cation species of the registered 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 systemic toxicity NOAEL for F0 males: 1.868 x 1.6 = 2.80 mg/kg bw/day

The systemic toxicity NOAEL for F1 males: 1.868 x 1.5 = 2.98 mg/kg bw/day

The systemic toxicity NOAEL for F0/F1 females: 1.868 x 1.7 = 3.18 mg/kg bw/day

The reproductive toxicity NOAEL for F0 males: 1.868 x 38.7 = 72.29 mg/kg bw/day

The reproductive toxicity NOAEL for F1 males: 1.868 x 34.7 = 64.82 mg/kg bw/day

The reproductive toxicity NOAEL for F0 females: 1.868 x 40.4 = 75.47 mg/kg bw/day

The reproductive toxicity NOAEL for F1 females: 1.868 x 35.7 = 66.68 mg/kg bw/day

The NOAEL for F1/F2 pups: 1.868 x 8.4 = 15.69 mg/kg bw/day

Table 1. Intergroup comparison of parental body weight gain (g) (pre-mating, selected time points)

	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)

Table 2. Intergroup comparison of body weight (g) during pregnancy

	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*

* 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. Intergroup comparison of body weight (g) during lactation

	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**

* 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 parental food consumption (g/rat/day) (pre-mating, selected time points) 

	Dietary Concentration of the test substance ion (ppm)
	Males				Females
Week	0	16	80	400/240	0	16	80	400/240
F0 1	19.9	20.1	19.4	14.4**	17.8	18.1	17.4	14.0**
4	29.9	30.5	29.9	25.3**	20.6	20.4	20.5	18.6**
8	30.4	30.5	30.0	26.4**	20.2	20.0	20.3	18.3**
Total (1-12)	2396.4	2436.1	2385.1	2050.0**	1683.3	1673.9	1664.7	1521.2**
F1 1	25.0	24.9	24.3	15.0**	21.0	20.7	20.5	13.9**
4	32.8	32.0	32.0	28.7**	22.9	22.4	22.2	22.1
8	31.2	30.9	30.6	29.2**	21.9	21.7	21.3	20.8*
Total (1-12)	2357.2	2324.9	2308.3	2048.1**	1694.3	1662.1	1648.0	1521.6**
* 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 5. Intergroup comparison of parental food utilisation (g growth/100 g food) (pre-mating, selected time points)   Dietary Concentration of the test substance ion (ppm)   Males Females Weeks 0 16 80 400 0 16 80 400/240 F0 Weeks 9-12   7.78   8.07   7.94   8.64*   4.06   4.47   4.59   4.49 Total (1-12) 16.41 16.41 16.59 16.32 10.99 11.13 11.28 11.00 F1 1-4   24.49   24.49   24.67   27.98**   16.03   15.58   16.88   20.93** 5-8 11.45 11.85 12.02 14.11** 6.37 6.87 6.91 8.49** 9-11 7.22 7.41 6.67 8.33* 3.89 3.57 3.72 4.48 Total (1-12) 14.86 15.03 14.98 16.65** 9.25 9.18 9.74* 11.44** * 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 6. Overall mean dose received (mg ion/kg/day).

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

# Approximately 4 weeks after selection of the F₁parents the dose level was reduced from 400 to 240 ppm test substance

Table 7. F0 Reproductive Performance: Litter A

Observation	Dietary Concentration of the test substance ion (ppm)
	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

 

Table 8. F1 Reproductive Performance: Litter A

Observation	Dietary Concentration of the test substance ion (ppm)
	0	16	80	240
Males: (proportion of fertile animals) %	93	93	93	96
Females: (proportion of fertile animals) %	97	97	100	100
Mean precoital interval (days)	3.50	3.10	2.56	2.76
Females placed with males	29	29	30	28
Pregnant	28	28	30	28
Median gestation interval (days)	22.0	22.0	22.1	22.0
Number of litters with all pups live born	20	22	23	18
Number of litters with all pups surviving to day 22	10	20	11	16

 

Table 9. Intergroup comparison of mean litter size

	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)

 

Table 10. Intergroup comparison of selected clinical observations in offspring

	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

 

Table 11. Litter parameters for F1a and F2a

Observation	Dietary Concentration of the test substance ion (ppm)
	0	16	80	400/240
F1A litter % all pups born live	98.6	97.0	98.7	96.8
F2A litter % all pups born live	96.4	95.5	97.0	95.6
F1A litter % all pups surviving to day 22	91.1	90.3	91.3	90.2
F2A litter % all pups surviving to day 22	83.0	94.0	86.9	88.3
F1A litter Mean litter size Day 1	10.8	10.6	11.0	9.2*
Day 5	9.9	9.7	10.1	8.4*
Day 11	9.8	9.6	10.0	8.3
Day 22	9.8	9.6	10.0	8.2*
Day 36	9.6	9.5	9.9	8.1*
F2A litter Mean litter size Day 1	10.5	11.1	10.7	10.8
Day 5	9.0	10.7*	9.4	9.5
Day 11	8.8	10.6*	9.2	9.4
Day 22	8.8	10.5*	9.2	9.4
Day 36	8.8	10.4*	9.2	9.3

*Statistically different from control, p<0.05

Table 12. Intergroup comparison of pup body weight gain (g) (selected time points)

	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)

Applicant's summary and conclusion

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).