Paraquat-dichloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

- Oral: reported systemic NOAEL = 1.25 mg/kg bw/day (cation) , recalculated systemic NOAEL = 1.7 mg/kg bw/day (pure test substance); reported reproductive toxicity NOAEL >= 7.5 mg/kg bw/day (cation), recalculated reproductive toxicity NOAEL >= 10.4 mg/kg bw/day (pure test substance); reported developmental NOAEL >= 7.5 mg/kg bw/day (cation), recalculated developmental toxicity NOAEL >= 10.4 mg/kg bw/day (pure test substance); male/female; Alderley Park rats; GLP compliant, study well documented, meets generally accepted scientific principles, acceptable for assessment; Lindsay 1982.

Link to relevant study records

Reference

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Dec 1979 to 28 Nov 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test:
Groups of 15 male and 30 female (F0 Parents) Wistar-derived rats were fed diets containing 0, 25, 75 and 150 ppm test substance. Test diets were fed experimental diets continuously throughout the study. After twelve weeks, animals were mated to produce the first (F1A) litter and subsequently re-mated to produce a second (F1B) litter. The breeding programme was repeated with F1 parents selected from the F1B offspring and F2 parents selected from the F2B offspring. Male parents were sacrificed after the last litters in each generation were produced. Female parents were sacrificed after the last litter of each generation was weaned. All parent animals of all generations were examined post mortem. For all generations (F1, F2 and F3), 50% of the offspring was examined in gross pathology and at least one pup per litter was subjected to a full post mortem histopathological examination.

- Parameters analysed / observed:
Clinical signs, body weight, food consumption, urinary test substance content, reproductive performance, gross pathology.

- Deviations from the original study design can be found in 'Any other information on materials and methods incl. tables'.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alderley Park

Remarks:

Wistar-derived

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P0) 29 days; (P1) 35 days; (P2) 35 days
- Weight at study initiation (treatment group averages): (P0) Males: 76.5 to 80.4 g; Females: 72.6 to 73.3 g; (P1) Males: 119.5 to 122.9 g; Females: 103.6 to 109.9 g ; (P2) Males: 120.2 to 127.0 g; Females: 106.5 to 109.7 g
- Housing: On arrival the rats were housed by sex in litters. Following randomisation and during the pre-mating period of the study they were housed, two females or one male per cage, 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 1.27 cm centres. The cages were suspended over collecting trays lined with absorbent paper. Attached to the front of each cage was a removable food hopper holding 400 g of food and the facility for a water bottle of 225 mL capacity. Attached to each cage was a card uniquely identifying the contained animals. At approximately day 14 of pregnancy, the cages housing the females were fitted with solid floors and supplied with autoclaved paper bedding material. The females remained in these cages throughout gestation and lactation.
- Diet: ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 25
- Humidity (%): 38 to 60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (7a.m. to 7 p.m.)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The appropriate volumes of a 1% w/w aqueous solution of the test substance were mixed with the base diet to give the desired concentrations of test substance in each diet. Water was added at 12.5% to 17.5% w/w of total diet to the mix which was then mixed in a mixer for 15 minutes. The final mix was passed through a pellet mill, which produced pellets of 16 mm diameter and variable length. The control diets were prepared in the same manner but without the addition of the test substance. The pelleted diets were dried in a hot air fan oven at a temperature not exceeding 50 °C for 2.5 hours to 5 hours.
- Rate of preparation of diet: weekly

Details on mating procedure:

- M/F ratio per cage: 1 / 2
- Length of cohabitation: until proof of mating
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: seperately

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aqueous extracts of diet were deproteinised with trichloroacetic acid and then centrifuged. The supernatant liquid was passed through a column containing an ion-exchange resin on which the test substance was absorbed and concentrated. The columns were washed, and then the test substance was eluted from the ion-exchange resin with ammonium chloride solution. Test substance was determined by reduction of a portion of the eluate with an alkaline dithionite reagent, and the absorbance of the coloured free-radical ion measured at 600 nm.

Duration of treatment / exposure:

For parent animals (P0, P1, P2): 12 weeks premating period (P0) or 11 weeks premating period (P1, P2), during mating period (max 10 days, if not successful an additional 10 days). Male animals were sacrificed after the last B-litters in each generation were produced; maternal animals were sacrificed after the last B-litter of each generation was weaned.

For offspring animals (F1AB, F2AB, F3AB): during weaning period 21 to 28 days, via food till parent selection day at 35 days post partum.

Frequency of treatment:

Continously

Details on study schedule:

Animals of the P0 generation were maintained on their respective diets for 12 weeks prior to mating. The animals were then mated on a one male to two female basis for a period until there was proof of mating (smears with sperm). Resulting litters (F1A) were sacrificed, examined macroscopically and then discarded. Shortly following the weaning of the F1A litters, the P0 generation were remated. Resulting litters (F1B) were reared when 15 males and 30 females were selected from each group to form the basis of the F1B generation. The P0 parents were examined post mortem and 5 males and 5 females from the F1B generation were subjected to a full post mortem examination and the tissues specified in Table 1 in 'Any other information on materials and methods incl. tables' were submitted for histopathological examination.

The P1 animals, selected 35 days post partum from F1B generation, were maintained on their respective diets for 11 weeks and were then mated on a one male to two female basis for a period until there was proof of mating (smears with sperm). Resulting F2A litters were sacrificed, examined macroscopically and then discarded. Shortly following the weaning of the F2A litters, the P1 generation were remated. P1 parents were sacrificed and examined macroscopically after weaning of the litters. The P1 parents were examined post mortem and 5 males and 5 females from the F2B generation were subjected to a full post mortem examination and the tissues specified in Table 1 in 'Any other information on materials and methods incl. tables' were submitted for histopathological examination.

After 35 days, 15 male and 30 female pups from each group were retained from as many litters as possible at weaning of the F2B generation and used as P2 generation. These animals were reared on their respective diets to an age of at least 11 weeks when they were mated on a one male to two females. Resulting litters (F3A) were sacrificed, examined macroscopically and discarded. Shortly following the weaning of the F3A litters the P2 animals were remated. Resulting litters (F3B) were reared to 35 days post partum when 10 males and 10 females were selected from each group for detailed macroscopic examination and organ weight analysis. Representative tissues were retained in fixative. Parent P2 animals were sacrificed and examined macroscopically.

Dose / conc.:

25 ppm

Remarks:

Dietary concentration of test substance cation; equivalent to 1.25 mg/kg bw/day.

Dose / conc.:

75 ppm

Remarks:

Dietary concentration of test substance cation; equivalent to 3.75 mg/kg bw/day.

Dose / conc.:

150 ppm

Remarks:

Dietary concentration of test substance cation; equivalent to 7.5 mg/kg bw/day.

No. of animals per sex per dose:

Control: 30 females, 15 males (plus additional 4 females, 2 males as microbiological sentinels)
Treatment: 30 females, 15 males (plus additional 4 females, 2 males as microbiological sentinels for the highest treatment group)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment: The appropriate number of parents per group for the second generation were selected from the F1B or F2B litter. The selection procedure was carried out as follows: The mean litter size per group was calculated from the Day 10 post partum litter information, available prior to the first date of selection. The litters used for selection were within ±3 of the mean litter size. At Day 35 post partum the average male and average female pup weights were calculated for each of the chosen litters. The pups nearest the average weight for each sex in the litter were selected and earmarked with the appropriate number. A maximum of two female pups and one male pup were chosen from any one litter, to become P1 or P2. The selected animals were housed according to the rack design. Records were kept of the parentage of the selected animals.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the study all rats were observed daily for abnormalities in clinical condition, behaviour and mortality. Any abnormalities were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly a detailed examination of each rat was made. Any abnormalities were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all rats were recorded at weekly intervals throughout the premating periods. The initial weights for the P0 parents were recorded immediately before first feeding experimental diets and the initial weights for the P1 and P2 parents were recorded at selection.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food Consumption for each cage of rats was recorded at weekly intervals throughout the premating periods. The food consumption was calculated, taking into account the daily food wastage for each cage. The food utilisation value per cage was calculated as the total food consumed divided by the total weight gained by the animal(s) in the cage during the period.

URINARY test substance content
- Individual urine samples were taken from three males and three females per group during the premating periods. The samples were collected over a 3 to 4 hour period during week 8 for the P0 parents and at Weeks 7 to 10 for the P1 and P2 parents (depending on the date of selection). During the collection period the rats were deprived of food but received water ad libitum. The urine samples collected were pooled by sex and group before submission for analysis of test substance content. The samples collected from each generation were from unrelated animals.

PARENT PATHOLOGY
A full post mortem examination was carried out on any rat found dead or moribund during the study and all tissues were processed for histopathological examination.

The moribund rats and all rats at termination were killed by inhalation of an overdose of halothane BP. All tissues were fixed in 10% formal saline, except eyes which were fixed in Davidson's solution and skin abnormalities which were fixed in Bouin’s solution. All submitted tissues were trimmed, processed end embedded in paraffin wax. Five micron thick sections were cut and stained with haematoxylin and eosin. Special stains were used at the discretion of the study pathologist.

Any female with imperforate vagina was killed after the premating period and subjected to a gross post mortem examination. Any abnormal tissues were submitted for histopathological examination.

Any male suspected of infertility, after pairing with a female for the A litter, was killed at termination of the generation and the reproductive tract submitted for histopathological examination.

Females suspected of infertility (ie no positive smear and no weight gain), after pairing for the A-litter, were killed after the supposed gestation period and the reproductive tract submitted for histopathological examination. However, where the number of parent females available for second litters was low, the suspected infertile females were remated with proven males in an attempt to produce sufficient B-litters. Some females used in this way produced normal B-litters and were treated as normal animals at termination. At termination all animals which failed to produce a B-litter. including those producing normal A-litters, received an autopsy and the reproductive tract, together with any abnormalities was submitted for histopathological examination. In subsequent discussion the terms sub-fertile and infertile refer respectively to females producing one litter only and females producing no litters.

Any female apparently pregnant (ie positive smear and weight gain) which failed to litter, was killed after the expected date of parturition and the uterus examined for implantation sites. If none were visible the staining technique of Salewski (1964) was used. From these females a small section of one uterine horn (removed prior to the Salewski test), the ovaries, cervix and any abnormalities were submitted for histopathological examination.

All surviving male parents from each generation were killed after completion of mating to produce the B-litters and all surviving female parents were killed after weaning the B-litter.

The P0 and P2 parents were subjected to gross post mortem examination at termination of the generation and the testes from all male rats, lungs from 8 male and 8 female rats per group, and any grossly abnormal tissues from all rats were submitted for histopathological examination.

At termination of the P1 parents twenty-five females and ten males in each group were subjected to a full post mortem examination and all tissues were either submitted for histopathological examination or stored in 10% formal saline. The remaining P1 parents were subjected to a gross post mortem examination and only testes and grossly abnormal tissues were submitted for histopathological examination.

Sperm parameters (parental animals):

Parameters examined in [P0/P2] male parental generations:
- Testes were subjected to histopathological examination; spermatogenesis, gross and microscopic appearance of the reproductive tract.

Parameters examined in [P1] male parental generations:
- Testes of 10 males were subjected to histopathological examination; spermatogenesis, gross and microscopic appearance of the reproductive tract.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 35 post partum
- maximum of 2 female pups/litter, 1 male pup/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,
For the A-litters of each generation, a gross post mortem examination 50% of the animals was performed, the rest of the litter was discarded after clinical examination.
For the B-litters of each generation, a full post mortem examination was performed on a designated number of pups (for F1 / F2: 5 males, 5 females; for F3: 10 males, 10 females). Gross post mortem examinations were done on 50% of the litter, the remaining pups were discarded after clinical examination.

GROSS EXAMINATION OF DEAD PUPS:
yes, for soft tissue examination

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the last B-litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last B-litter of each generation was weaned.
- The moribund rats and all rats at termination were killed by inhalation of an overdose of halothane BP

HISTOPATHOLOGY / ORGAN WEIGTHS & GROSS NECROPSY
The tissues indicated in Table 'Tissues submitted at post mortem examinations' in the field 'Any other information on materials and methods incl. tables' were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 35 days of age.
- 50% of these animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

HISTOPATHOLOGY / ORGAN WEIGTHS & GROSS NECROPSY
After selection of the next generation parents, 5 male and 5 female rats per group from the F1B and F2B litters and ten male and ten female rats per group from the F3B litters were subjected to a full post mortem examination and the tissues submitted for histopathological examination. Approximately 50% of the remaining rats were subjected to a gross post mortem examination with only abnormal tissues submitted for examination. The remainder were discarded after clinical examination.

The tissues indicated in Table 'Tissues submitted at post mortem examinations' in the field 'Any other information on materials and methods incl. tables' were prepared for microscopic examination and weighed, respectively.

Statistics:

Weekly body weight gain and food consumption and total food consumption were considered by analysis of variance separately for each sex.
Food utilisation was also considered by analysis of variance, separately for each sex, over specific periods of the study. For the P0 parents food utilisation was considered for the periods weeks 1-4, 5-8, 9-12 and 1-12, for the P1 parents the periods weeks 1-3, 6-11 and 1-11 were considered. The reason for excluding weeks 4 and 5 for the P1 generation was that no food consumption measurements were available for any top dose group cage for both week 4 and week 5, therefore no estimate of food utilisation was possible over this period for this group. This can be seen in the food utilisation for weeks 1-11, where this parameter has been excluded for the 150 ppm group. For the P2 parents food utilisation was considered for the periods weeks 1-4, 5-8, 9-11 and 1-11.
All analyses allowed for the replicate design of the study. Whenever there were any missing values the group means were adjusted for replicate before comparisons were made. Treatment group means were compared with the control group mean using a two sided Student’s t-test based on the relevant mean square from the analysis.
The data were checked, using simple plats, for any extreme values. Where such values were detected, their influence on the conclusions was determined by repeat analysis omitting these values.

Reproductive indices:

The fertility or otherwise of each male and female was established by the success (as measured by the production of a viable litter) of each mating. Where it was not possible to establish whether individual animals were fertile or infertile, these animals were excluded from consideration in the fertility indices. Treatment group means were compared with the control group mean using a one-sided Fishers exact test.
The mean gestation period, (measured in days from date of the positive smear to date of birth) for each treatment group was compared with the control group using a two-sided Student's t-test.

Offspring viability indices:

The proportion of pups born alive (Live Born Index) and the proportion of viable pups which survived to day 21 (Survival Index) calculated individually for each litter were considered by analysis of variance following transformation using the double arcsine transformation. Similarly, total litter size at days 0, 4, 10, 21 and 28 and initial litter weight and litter weight gain at days 4, 10, 21 and 28 (separately for male and female pups) were considered by analysis of variance. In all the above analyses treatment group means were compared to the control group mean using a two-sided Student's t-test based on the relevant mean square from the analysis.
The proportion of viable litters in which all pups died by day 10 (Maternal Neglect Index) was also analysed comparing each treated group with the control group using a one-sided Fisher’s exact test.
Weekly body weight gain during pregnancy was considered by analysis of covariance with adjustment being made for the effect of remating. Treatment group means were then compared with the control group mean using a two-sided Student’s t-test based on the relevant mean square the analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Parent rats generally remained in good condition throughout the study. The only frequently observed condition was hair loss in female rats but this was not treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

12 animals died or became moribund during the study. Most casualties were females from the top dose group, which died after producing the B-litter.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no consistent evidence of an effect on female body weight gain. At week 2 the 150 ppm dose group showed a statistically significantly reduced bodyweight gain but this did not persist and there was no other indication of any treatment related effect.

In males there was a significantly reduced body weight gain throughout the study in the 75 ppm dose group, but there were no statistically significant effects in the other dose groups, therefore, the reduction was not considered to be a treatment related effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were a few instances of statistically significant increases and reductions in weekly food consumption of both males and females in the 150 ppm dose group and increases in the food consumption of females in the 25 ppm dose group.

In females there was no clear dose related effect but all treated groups showed a marginal increase in food consumption for most time points from week four which led to a significantly increased overall food consumption for animals in the 25 ppm dose group.

In males an increased food consumption was suggested in the 25 ppm and 150 ppm dose groups from week 5 onwards, but there was no evidence of an increase in the 75 ppm dose group.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food utilisation of females in all treatment groups tended to be higher throughout the study although this was never statistically significantly different from the control. In males food utilisation values were statistically significantly increased on two occasions (75 ppm dose group during weeks 1 to 4 and the 150 ppm dose group during weeks 9 to 12), however the overall food utilisation of the treatment groups was similar to the control value.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Before weaning: microscopic analysis of the lungs revealed severe generalised congestion with areas of alveolar oedema and hyaline membrane formation. Other changes consisted of perivascular oedema and mixed inflammatory cell infiltration and a variable alveolar exudate consisting mainly of large macrophages with a few neutrophils. All lungs also showed a few areas of pro-fibroblast proliferation with early alveolar fibrosis and epithelialisation. The microscopical changes were typical of severe lung injury.
- During weaning period of the F1B litters: two animals showed slightly more long-standing lesions, (severe subacute lung injury), in which there were extensive irregular areas of consolidation due to alveolar fibrosis accompanied by a dense exudate composed mainly of macrophages. In addition there was hypertrophy, hyperplasia and mucoid metaplasia of bronchial epithelium and alveolar epithelialisation. Remaining non-consolidated areas showed all the changes seen in acute lung injury.
- At termination: microscopically the lungs contained localised areas of consolidation due to alveolar fibrosis often with epithelialisation of the few alveoli remaining patent which sometimes contained a few macrophages and pro-fibroblasts. There was also hypertrophy, hyperplasia and mucoid metaplasia of bronchial epithelium with perivascular oedema and mixed inflammatory cell infiltration. These changes were discrete and localised. Remaining areas of lung parenchyma were normal.
- At termination: no lesions typical of the test substance were seen in females from any other treatment groups or in control females. However, some females in all groups including controls showed focal accumulation of large, foamy macrophages (focal alveolar histiocytosis) in sub-pleural and peribronchial tissue. Grossly these lesions were seen as small white or cream foci on the surface of the lung. In some lesions there was also cholesterol cleft formation, occasionally accompanied by alveolar epithelialisation and mononuclear cell infiltration of alveolar walls. The incidence and severity of the lesion appeared to be related to treatment. Although present in all animals which showed chronic lung injury the severity of focal alveolar histiocytosis did not seem to be increased when co-existent with typical lung damage. There was a small pulmonary adenoma in one animal receiving 150 ppm test substance, but this was an isolated finding and considered incidental to treatment.
- At termination: on examination of the reproductive tract of sub-fertile and infertile females, in most cases no reason could be found for the poor reproductive performance. The few lesions in the reproductive tract were isolated incidental findings of no toxicological significance.
- At termination: examination of the P0 males the only significant change was in the lungs. There were no lesions typical of lung injury but there was a dose-related increase in the incidence and severity of focal alveolar histiocytosis in males receiving 75 and 150 ppm test substance.
- At termination: There were no changes of toxicological significance in the reproductive tract either of males suspected of infertility or of those with a normal breeding record.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

URINARY TEST SUBSTANCE
The results in terms of test substance excreted (μg/rat) and test substance concentration (μg/mL) in urine show that dose related absorption of the test substance took place in the treated groups during the study.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no adverse effects on male or female fertility during the production of the F1A or F1B litters. The incidence of sub-fertility and infertility was low and unrelated to treatment.

The body weight gain of pregnant rats was generally lower in the treated groups compared with the control group, however no statistically significant differences were observed.

There were no treatment related effects on gestation period for either the F1A or F1B litters and although statistically significant reductions were seen in the 75 ppm dose group for the F1A litter and the 25ppm dose group for the F1B litter, they were of no biological significance.

There were no treatment related effects on live born, maternal neglect, or survival indices or on litter size for either the F1A or F1B litters. However, in the F1B litter a slightly higher maternal neglect index and slightly lower survival index was seen in the control, 25 ppm and 75 ppm dose groups, when compared with the F1A, F2A and F2B litters. It is likely that these differences were caused by a noise disturbance which occurred during the production of the F1B litters and resulted in a number of whole litter losses in the afore-mentioned groups.

Dose descriptor:

NOAEL

Remarks:

reproductive performance, P0

Effect level:

>= 150 ppm

Based on:

test mat.

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 concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance, P0

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, P0

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 5.2 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, P0

Effect level:

5.2 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

mortality

gross pathology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Critical effects observed:

yes

Lowest effective dose / conc.:

150 ppm

System:

respiratory system: lower respiratory tract

Organ:

alveoli

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10.4 mg/kg bw/day (nominal)

System:

respiratory system: lower respiratory tract

Organ:

alveoli

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

For P1 Parents: rats generally remained in good condition throughout the study. The only frequently observed condition was hair loss in female rats but this was not treatment related.

For P2 parents: rats generally remained in good condition throughout the study. The only frequently observed condition was hair loss in female rats but this was not treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

For P1 parents: 18 animals died or became moribund during the study. Most casualties were females from the top dose group, which died after producing the A-litter.

For P2 parents: 8 animals died or became moribund during the study. Most casualties were females from the top dose group, but did not show any trend.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For P1 parents: There was no consistent evidence of any effect on body weight gain in either males or females.

For P2 parents: There was a significant increase in body weight gain in females from the 75 ppm test substance group throughout the premating period and in males from the 150 ppm test substance group during the latter half of the premating period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For P1 parents: Females from all treatment groups showed a decrease in food consumption from week 6 onwards with the exception of week 10 when all treatment group values were greater than control. The decreases were not dose related and only statistically significant in the top dose group at week 8. The males showed a similar decrease in food consumption from week 5 onwards, the only statistically significant reduction being in the top dose group at week 9.

For P2 parents: There was no evidence of any effect on food consumption at any dose level, in either sex.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

For P1 parents: Food utilisation in the treatment groups of both sexes generally tended to be better than the control group, although statistical significance was only achieved in males from the 75 ppm dose group during weeks 6 to 11.

For P2 parents: The food utilisation of the females was unaffected by treatment, but males from the 150 ppm dose group showed improved food utilisation when compared to the control group.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For P1 parents: during the study: Sixteen female parents and two male parents died or were killed during the study. The two male deaths were not treatment related, but 13 of the females were from the 150 ppm group and in the late stages of lactation. At necropsy of these mothers significant gross changes were confined to the lungs and were similar to those seen in the P0 parents dying intercurrently. No significant changes were seen in the reproductive organs of females dying as a result of lung damage.
At termination of the P1 females, the pathological findings of toxicological significance were confined to the lungs. Mild to severe chronic lung injury was present in 5 of the 17 females receiving 150 ppm test substance which survived to termination. One female which was in poor condition at termination showed severe sub-acute lung injury similar to that seen in animals dying intercurrently. No lesions characteristic of lung injury were seen in the two lower dose groups or in control animals.

For P2 parents: 2 control males and six females receiving 150 ppm test substance died or were killed in extremis. All the females showed lesions characteristic of severe lung injury, similar to those described in the two previous generations.
At termination of the P2 parents significant changes were again confined to the lungs. They consisted of sub-acute or chronic lung injury in females receiving 150 ppm of the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

For P1 parents: during weaning: 13 females died during weaning. Microscopically, all showed typical severe lesions of acute or sub-acute lung injury as described in the P0 females. Increased siderophage formation in the spleen was the only other change with an increased incidence in females dying intercurrently from lung injury. 5 out of 13 (38%) animals, were affected compared with only 1 at termination. This change is not thought to be a direct toxic effect of the test substance. It is more likely to be an indication of increased red cell turnover in response to changes in oxygen tension in the blood caused by severe dyspnoea.
- At termination: the incidence and severity of focal alveolar histiocytosis was significantly increased in the group receiving 75 and 150 ppm dose groups but not in animals receiving 25 ppm.
- At termination: there were no changes of toxicological significance in the reproductive tract of any P1 female at termination. The few lesions found in the reproductive tract of sub-fertile or infertile P1 females were of no toxicological significance.
- At termination: lesions in all other organs were of a minor nature and typical of the spontaneous and age-related changes seen in rats of this age and strain. The incidence of all changes was unrelated to treatment.
- At termination of the P1 males the only change of toxicological significance occurred in the lungs, where there was a definite dose related increase in the incidence and severity of focal alveolar histiocytosis in animals receiving 75 and 150 ppm test substance. However, there were no lesions characteristic of lung injury. A few animals in control and treated groups were suspected of infertility. In all these animals, spermatogenesis appeared normal and the gross and microscopic appearance of the reproductive tract was unremarkable except in one male receiving 150 ppm test substance where there was severe purulent inflammation of the preputial gland. Spermatogenesis was, however, normal in this animal, and as it was an isolated finding, it is not considered to be of any toxicological significance.
- At termination: the testes of fertile males were normal except for one animal receiving 150 ppm test substance which showed marked atrophy of seminal tubules in one testis only. Tubular atrophy is a common age related change in the rat and this is considered to be an incidental finding.
- At termination: there was also a slightly increased incidence of minimal or mild prostatitis in males receiving 150 ppm test substance. The change was very minor involving only a few prostatic acini and fertility was not affected in any animal. Prostatitis of this degree is a common lesion in laboratory rats and the slightly higher incidence in the top dose group is not considered to be of toxicological significance.

For P2 parents: 2 control males and six females receiving 150 ppm test substance died or were killed in extremis. 2 of these females showed centrilobular hepatic necrosis. This was probably caused by severe hypoxia secondary to lung damage and is considered unlikely to be due to a primary hepatotoxic effect of the test substance.
- At termination of the P2 parents significant changes were again confined to the lungs. They consisted of sub-acute or chronic lung injury in females receiving 150 ppm test substance and a dose related increase in the incidence of focal alveolar histiocytosis in females receiving 75 ppm and 150 ppm test substance and in males receiving 150 ppm test substance.
- At termination: there were no other treatment related changes in the lungs. There were no changes of toxicological significance in the reproductive tract of P2 parents at termination. The few lesions detected were isolated incidental findings and the incidence of infertility was low and unrelated to treatment.
 

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

URINARY TEST SUBSTANCE
For P1 parents: the results in terms of test substance excreted (μg/rat) and test substance concentration (μg/mL) in urine show that dose related absorption of the test substance took place in the treated groups during the study.

For P2 parents: the results in terms of test substance excreted (μg/rat) and test substance concentration (μg/mL) in urine show that dose related absorption of the test substance took place in the treated groups during the study.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

For P1 parents: in all these animals, spermatogenesis appeared normal and the gross and microscopic appearance of the reproductive tract was unremarkable except in one male receiving 150 ppm test substance where there was severe purulent inflammation of the preputial gland. Spermatogenesis was, however, normal in this animal, and as it was an isolated finding, it is not considered to be of any toxicological significance.

Reproductive performance:

no effects observed

Description (incidence and severity):

For P1 parents: there was some indication of reduced fertility in the 25 ppm and 75 ppm dose group females during the production of the F2A and F2B litters. The reduction was slight, did not achieve statistical significance and was not supported by any effect on the top dose group. It was not therefore considered to be related to treatment.

For P2 parents: There were no adverse effects on male and female fertility during the production of the F3A and F3B litters.
The bodyweight gain of pregnant P2 females from the 75 ppm dose group was increased compared to the control group during the production of the F3A and F3B litters and this attained occasional statistical significance. However as there was no evidence of any treatment related effect in the other dose groups this increase is not considered to be of biological significance. There were no adverse effects on the gestation period, live born, maternal neglect, or survival indices or on litter size for the F3A or F3B litters.

For P1 parents: the body weight gain of pregnant P1 females during the production of the F1B litter was generally lower in the treated groups compared with the control group, although no statistical significance was achieved. This effect was similar to that seen in the F0 generation. There were no consistent or dose related effects on gestation period for either the F2A or F2B litter and although a statically significant increase in gestation period was seen in the 25 ppm dose group for the F2A litter, this was an isolated finding and not considered to be treatment related. There were no adverse effects on live born, maternal neglect or survival indices or on litter size for the F2A or F2B litters.

Dose descriptor:

NOAEL

Remarks:

reproductive performance, P1 and P2

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproduction

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance, P1 and P2

Effect level:

>= 10.4 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproduction

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, P1 and P2

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 1.25 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Effect level:

1.7 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Critical effects observed:

yes

Lowest effective dose / conc.:

75 ppm

System:

respiratory system: lower respiratory tract

Organ:

alveoli

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5.2 mg/kg bw/day (nominal)

System:

respiratory system: lower respiratory tract

Organ:

alveoli

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

In general the F1 offspring appeared healthy throughout the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Mortality in the F1B litter was slightly higher than for the other litters. This appeared to be the result of slightly increased maternal neglect due to a noise disturbance at the time of littering.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1A and F1B litters: there was evidence of a reduction in the litter weight gain of both male and female pups in both the F1A and the F1B litters with instances of statistically significant reductions mainly in the 25 ppm and 75 ppm dose groups. The effect was not dose-related.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross abnormalities of any kind were seen in male or female reproductive organs and the development of the genital tract was within normal limits all four groups of animals. At termination of the F1B offspring the only change of possible toxicological significance occurred in the lungs of pups receiving 150 ppm test substance. In 4 out of 5 males and 2 out of 6 females there was mild perivascular inflammatory cell infiltration in the lungs.

Histopathological findings:

no effects observed

Description (incidence and severity):

At termination of the F1A offspring a small number of macroscopically abnormal organs were examined histopathologically, but there were no changes of toxicological significance.

Unilateral necrosis of the testis and epididymis was present in one male pup receiving 150 ppm test substance. This was an isolated incidental finding and not considered to be of toxicological significance. There were no significant abnormalities in the reproductive organs. Development of the genital tract was within normal limits in all animals. There were no changes of toxicological significance in any other organs.

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity, F1

Generation:

other: F1A/F1B

Effect level:

>= 150 ppm

Based on:

other: test material cation

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity, F1

Generation:

other: F1A/F1B

Effect level:

>= 10.4 mg/kg bw/day (nominal)

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F1

Generation:

other: F1A/F1B

Effect level:

>= 150 ppm

Based on:

other: test material cation

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F1

Generation:

other: F1A/F1B

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

For F2 pups: offspring appeared healthy throughout the lactation period.
For F3 pups: offspring appeared healthy throughout the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For F2 pups: The majority of offspring losses during lactation were made up of pups which were missing and presumed cannibalised, however any pups found dead or moribund were examined for possible cause of death.

For F3 pups: The majority of offspring losses during lactation were made up of pups which were missing and presumed cannibalised, however any pups found dead or moribund were examined for possible cause of death. Mortality was lowest in the F3 offspring.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For F2 pups: there were no effects on litter weight gains in either sex for the F2A or F2B litters.

For F3 pups: There were no effects on the litter weight gains of the F3A female pups, however instances of statistically significantly reduced litter weight gains were seen in males from the 25 ppm dose group. The litter weight gains of F3B male and female pups in the 25 ppm and 150 ppm dose groups were generally lower but not statistically significantly different from their respective control group. The above effects showed no relationship to dose and were considered to be of no biological significance.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For F2 pups: no treatment related effects were found. At termination of the F2A offspring there were no changes of toxicological significance and development of the reproductive organs appeared normal. The non-treatment related findings consisted of two F2A pups from a litter in the 25 ppm group showed evidence of internal bleeding and subarachnoid haemorrhage. In the F2B litters one female pup from the control group had a cleft palate and enlarged lateral ventricles of the brain, and a pup from a top dose litter had no fourth digit on either fore limb.

For F3 pups: no treatment related effects were found. At termination of the F3A offspring there were no changes of toxicological significance.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

For F2 pups: no treatment related effects were found. At termination of the F2B offspring a few treated males (1 or 2 in each dose group) showed minimal to moderate atrophy of seminal tubules. No control animals were affected. As the incidence was so low it is considered unlikely that this finding had any toxicological significance. One male pup receiving 150 ppm test substance was found to have marked internal hydrocephalus. Testicular hypoplasia and hyposecretion in the secondary sex organs in this animal were probably related to abnormal hypothalamic function. This was an isolated finding and not considered to be of any toxicological significance. Hydrocephalus is a spontaneous congenital anomaly seen occasionally in the Wistar-derived rat. Otherwise development of the reproductive tract was normal in all animals of both sexes.

For F3 pups:At termination of the F3B offspring no treatment related changes were detected. Testicular hypoplasia in one male receiving 150 ppm test substance was considered to be an isolated incidental finding. Development of the genital tract was within normal limits in all other animals.

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity, F2

Generation:

other: F2A/F2B

Effect level:

>= 150 ppm

Based on:

other: test material cation

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity, F2

Generation:

other: F2A/F2B

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F2

Generation:

other: F2A/F2B

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F2

Generation:

other: F2A/F2B

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

developmental toxicity, F3

Generation:

other: F3A/F3B

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity, F3

Generation:

other: F3A/F3B

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F3

Generation:

other: F3A/F3B

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 7.5 mg test substance cation/kg bw/day. Recalculated effect level for the pure test substance can be found in 'Any other information on results incl. tables'.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity, F3

Generation:

other: F3A/F3B

Effect level:

>= 10.4 mg/kg bw/day

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Remarks on result:

other:

Remarks:

Recalculated value of the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 1.25 mg test substance cation / kg bw = 1.7 mg pure pure test substance/ kg bw

(100/72.4) x 3.75 mg test substance cation / kg bw = 5.2 mg pure pure test substance/ kg bw

(100/72.4) x 7.5 mg test substance cation / kg bw = 10.4 mg pure pure test substance/ kg bw

Conclusions:

It was concluded that the test substance had no effect on reproductive performance or development of the reproductive organs of Alderley Park rats when administered at dietary levels up to 150 ppm over 3 generations. Increased incidences of lung lesions at 150 and 75 ppm indicate that the overall NOAEL is 25 ppm test substance, equivalent to approximately 1.7 mg pure test substance/kg bw/day.

Executive summary:

The potential reproductive effects of the test substance were investigated in a 3-generation (2 litters per generation) study. Groups of 15 male and 30 female (P0 parents) weanling Alpk Wistar-derived rats were fed diets containing 0, 25, 75, or 150 ppm (dietary equivalent to 0, 1.25, 3.75 and 7.5 mg test substance cation/kg bw/day). Test diets were fed continuously throughout the study. Samples of diet were analysed for achieved concentration and homogeneity. After 12 weeks pre-mating administration of diets, the P0 animals were mated to produce the first litters, F1a and F1b.These litters were reared to weaning. The breeding programme was repeated with P1 selected from the F1b litters which were mated at least 11 weeks after selection giving F2a and F2b litters. The P2 to P3 mating was identical to the P1 to P2 mating. During mating two females were housed with one male. Daily vaginal smear examinations were performed to determine when mating occurred – designated Day 1 of gestation. After 21 days if there was no evidence of mating, the first male was removed and after a 3 day interval was replaced by a male of proven fertility. During the study all rats were observed daily for mortality, abnormalities in clinical condition and behaviour. Body weight and food consumption were determined regularly. Urine samples were collected pre-mating from 3 parental animals/sex/ group for test substance determinations. Moribund or dead rats were examined post mortem. Parental animals were fully examined post mortem at termination of the generation and selected tissues (lung, testes and any organs with abnormal appearance) were examined histopathologically. Twenty five female and 10 male P1 animals were examined in detail post mortem and a wide range of tissues were examined histopathologically. All pups found dead or considered abnormal were given an extensive examination. Litters were examined at least once daily and grossly abnormal pups or those dying before day 18 were removed for teratological examination by Wilson sectioning. Approximately 50% of pups were killed post weaning and subjected to a gross post mortem examination with abnormal tissues taken for histopathological examination. Five pups/sex/group from the F1b and F2b litters and 10/sex/group from the F3b litters received a full examination post mortem with a wide range of tissues examined histopathologically. A count of all live and still-born pups was made within 24 hours of parturition (day 0) and thereafter at days 4, 10 and 21 post partum. The sexes and any clinical abnormalities of the pups were also recorded at these times. Individual pup body weights were recorded within 24 hours of birth (day 0) and at days 4, 10, 21 and 28 post partum. The mean gestation period, body weight gain during pregnancy, proportion of pups born alive, survival to day 21 and proportion of litters viable at day 10 were determined.

Dietary analyses showed achieved concentration, homogeneity and stability to be satisfactory. Urine analyses for the test substance showed a good dose relationship in P0 animals but in P1 males and P2 males and females the results for 75 ppm animals were similar to those from 150 ppm animals indicating saturation may have been approached.

There were no adverse effects on parental body weights or food consumption. However, there was a high incidence of mortality (27-43%) in the high dose P0, P1 and P2 females, mostly due to severe lung damage caused by the test substance. No treatment-related changes were found in the reproductive performance (male and female fertility, live-born and survival indexes, and litter size) or in the reproductive organs of parents or offspring. Body weight gain during pregnancy was reduced in the second mating of each generation in animals from the 150 ppm group although litter weight gain was satisfactory by day 28 in all instances. Development of the reproductive organs in all treated offspring was substantially comparable to that in controls.

Post mortem findings related to treatment were seen at 150 ppm and occasionally at 75 ppm. A red/purple discolouration of the lung and fibrosis were seen in both pups and parents from the 150 ppm groups. Alveolar histiocytosis was more pronounced at 150 and 75 ppm in all male parents and in P1 and P2 females. A high incidence of renal pelvis dilatation was evident in all groups including controls. An increased incidence of internal hydrocephalus was seen in F2b males. There was no evidence of eye lesions associated with test substance administration.

It was concluded that the test substance had no effect on reproductive performance or development of the reproductive organs of Alderley Park rats when administered at dietary levels up to 150 ppm over 3 generations. Increased incidences of lung lesions at 150 and 75 ppm indicate that the overall NOAEL is 25 ppm test substance cation, equivalent to approximately 1.7 mg pure test substance/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10.4 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP compliant, study well documented, meets generally accepted scientific principles, acceptable for assessment.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction - Rat

The potential reproductive effects of the test substance were investigated in a 3-generation (2 litters per generation) study. Groups of 15 male and 30 female (P0 parents) weanling Alpk Wistar-derived rats were fed diets containing 0, 25, 75, or 150 ppm (dietary equivalent to 0, 1.25, 5.2 and 7.5 mg test substance cation/kg bw/day). Test diets were fed continuously throughout the study. Samples of diet were analysed for achieved concentration and homogeneity. After 12 weeks pre-mating administration of diets, the P0 animals were mated to produce the first litters, F1A and F1B.These litters were reared to weaning. The breeding programme was repeated with P1 selected from the F1b litters which were mated at least 11 weeks after selection giving F2A and F2B litters. The P2 to P3 mating was identical to the P1 to P2 mating. During mating two females were housed with one male. Daily vaginal smear examinations were performed to determine when mating occurred – designated Day 1 of gestation. After 21 days if there was no evidence of mating, the first male was removed and after a 3 day interval was replaced by a male of proven fertility. During the study all rats were observed daily for mortality, abnormalities in clinical condition and behaviour. Body weight and food consumption were determined regularly. Urine samples were collected pre-mating from 3 parental animals/sex/ group for test substance determinations. Moribund or dead rats were examined post mortem. Parental animals were fully examined post mortem at termination of the generation and selected tissues (lung, testes and any organs with abnormal appearance) were examined histopathologically. Twenty five female and 10 male P1 animals were examined in detail post mortem and a wide range of tissues were examined histopathologically. All pups found dead or considered abnormal were given an extensive examination. Litters were examined at least once daily and grossly abnormal pups or those dying before day 18 were removed for teratological examination by Wilson sectioning. Approximately 50% of pups were killed post weaning and subjected to a gross post mortem examination with abnormal tissues taken for histopathological examination. Five pups/sex/group from the F1b and F2b litters and 10/sex/group from the F3b litters received a full examination post mortem with a wide range of tissues examined histopathologically. A count of all live and still-born pups was made within 24 hours of parturition (day 0) and thereafter at days 4, 10 and 21 post partum. The sexes and any clinical abnormalities of the pups were also recorded at these times. Individual pup body weights were recorded within 24 hours of birth (day 0) and at days 4, 10, 21 and 28 post partum. The mean gestation period, body weight gain during pregnancy, proportion of pups born alive, survival to day 21 and proportion of litters viable at day 10 were determined.

Dietary analyses showed achieved concentration, homogeneity and stability to be satisfactory. Urine analyses for the test substance showed a good dose relationship in P0 animals but in P1 males and P2 males and females the results for 75 ppm animals were similar to those from 150 ppm animals indicating saturation may have been approached.

There were no adverse effects on parental body weights or food consumption. However, there was a high incidence of mortality (27-43%) in the high dose P0, P1 and P2 females, mostly due to severe lung damage caused by the test substance. No treatment-related changes were found in the reproductive performance (male and female fertility, live-born and survival indexes, and litter size) or in the reproductive organs of parents or offspring. Body weight gain during pregnancy was reduced in the second mating of each generation in animals from the 150 ppm group although litter weight gain was satisfactory by day 28 in all instances. Development of the reproductive tract in all treated offspring was substantially comparable to that in controls.

Post mortem findings related to treatment were seen at 150 ppm and occasionally at 75 ppm. A red/purple discolouration of the lung and fibrosis were seen in both pups and parents from the 150 ppm groups. Alveolar histiocytosis was more pronounced at 150 and 75 ppm in all male parents and in P1 and P2 females. A high incidence of renal pelvis dilatation was evident in all groups including controls. An increased incidence of internal hydrocephalus was seen in F2b males. There was no evidence of eye lesions associated with test substance administration.

It was concluded that the test substance had no effect on reproductive performance or development of the reproductive organs of Alderley Park rats when administered at dietary levels up to 150 ppm over 3 generations. Increased incidences of lung lesions at 150 and 75 ppm indicate that the overall NOAEL is 25 ppm test substance cation, equivalent to approximately 1.7 mg pure test substance/kg bw/day.

Effects on developmental toxicity

Description of key information

- Oral: reported maternal NOAEL = 3 mg/kg bw/day (cation), recalculated maternal NOAEL = 4.1 mg/kg bw/day (pure test substance); reported developmental NOAEL >= 8 mg/kg bw/day (cation), recalculated developmental toxicity NOAEL >= 11.0 mg/kg bw/day (pure test substance); female; Alderley Park rats; GLP compliant, similar to OECD 414; Hodge 1992.

- Oral: reported maternal NOAEL = 15 mg/kg bw/day (cation), recalculated maternal NOAEL = 20.7 mg/kg bw/day (pure test substance); reported developmental NOAEL = 15 mg/kg bw/day (cation), recalculated developmental toxicity NOAEL = 20.7 mg/kg bw/day (pure test substance); female; CD-1 mice; GLP compliant, similar to OECD 414; Palmer 1992.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Jun 1992 to 19 Jun 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD-1(ICR)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: 25 to 29 g.
- Housing: During acclimatisation and mating the animals were housed in grid bottomed polypropylene cages over paper-lined trays. Males were housed individually and females in groups of up to five, except when paired for mating. Mated females were individually housed in solid bottomed polypropylene cages, with wood sawdust provided as bedding.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 11 days prior to mating.

DIET AND WATER QUALITY VERIFICATION
- Each batch of diet was delivered with an accompanying certificate of analysis detailing nutritional composition and levels of specified contaminants (heavy metals, aflatoxins and insecticides). The water is periodically analysed by independent analysts for microbiological purity and levels of halogenated hydrocarbons. There were no known contaminants in the food or water that were considered likely to interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 25
- Humidity (%): 39 to 68
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
3 Jun 1992 to 19 Jun 1992

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once for the study, prior to the start off dosing, and divided into appropriate aliquot portions. Dosing solutions were prepared with purified water. The weighed quantity of test material was dissolved in the appropriate volume of vehicle. Separate solutions were prepared, corrected for purity content, for each dose level. The aliquots were stored at room temperature protected from light, until required for dosing.

VEHICLE
- Amount of vehicle: 10 mL/kg bw calculated and adjusted daily on an individual body weight basis.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As a check on the accuracy of preparation, samples from each dosing solution were determined by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The method of analysis was validated in a previous study.

Details on mating procedure:

- Impregnation procedure: cohoused.
- M/F ratio per cage: 1/1.
- Length of cohabitation: Overnight on four consecutive days.
- Proof of pregnancy: vaginal plug (either in situ or in the cage tray) referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Days 6 to 15 of pregnancy inclusive.

Frequency of treatment:

Daily.

Duration of test:

Till day 18 of pregnancy.

Dose / conc.:

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

Remarks:

Group 2, low dose group. Concentration of test substance cation.

Dose / conc.:

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

Remarks:

Group 3, mid dose group. Concentration of test substance cation.

Dose / conc.:

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

Remarks:

Group 4, high dose group. Concentration of test substance cation.

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor, on the basis of results from a preliminary study performed.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily from day 0 of pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6 to 15, inclusive and 18 of pregnancy. The dead body weight of the killed animals was recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15 and 15 to 18 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 18 of pregnancy.
- Organs examined: major organs, lungs plus trachea, and kidneys were removed and weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of implantations in uterine horns classified as early resorptions/late resorptions/dead fetuses/live fetuses.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter (half fixed in Bouin’s fluid, the rest in 70% alcohol)
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

The following parameters were analysed by analysis of variance: maternal body weight, maternal body weight gain, maternal food consumption, maternal organ weights, maternal body weight-related organ weights, the numbers of implantations and live fetuses per female, percentage post-implantation loss, percentage of male fetuses, litter weight and mean fetal weight.
Fetal data was analysed on litter basis. Percentages were transformed before analysis using the double arcsine transformation.
Each treatment group was compared to the control group using Student's t-test, based on the error mean square from the analysis of variance. The tests were two-sided and statistical significance was set at p < 0.05 and 0.01.
Fisher's exact probability test was used to compare each treated group with the control group for the following: the proportion of foetuses with each individual defect, the proportion of litters with each individual defect
The tests were one-sided and statistical significance was set at p < 0.05 and 0.01 .

Indices:

percentage post implantation loss and percentage of male foetuses.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The animals which were terminated prematurely showed prior to death piloerection, laboured respiration, hunched posture, were hypothermic, hypoactive and/or had pale extremities and eyes. There were no other premature deaths and no other treatment-related changes in clinical conditions were observed in other treatment groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the group treated at 25 mg/kg bw/day, one female was found dead on day 16 of pregnancy. There were no changes in clinical condition observed prior to death. Four other females in this dose group were terminated prematurely, due to poor clinical condition at the end of the dosing period/beginning of the post dosing period (days 16, 16, 17 and 15 of pregnancy, respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an effect of treatment at 25 mg/kg bw/day on maternal body weight gain. A statistically significant (p < 0.01) retardation of maternal body weight gain over days 12 to 15 and 15 to 18, and also over the entire dosing period (days 6 to 15) and the entire pregnancy period (days 0 to 18) was seen in comparison with the control group. Body weight gain of this group from day 0 to day 18 after adjustment for gravid uterus weight was also slightly retarded in comparison with the control group, but statistical significance was not achieved. Body weight itself in this group was significantly lower than the control group on day 15 (p < 0.01) and day 18 (p < 0.01) of pregnancy (Table 1 and 2 in 'Any other information on results incl. tables').
At 7.5 or 15 mg/kg bw/day, maternal body weight gain was unaffected by treatment and body weights and adjusted body weight gains were similar to the control group, throughout pregnancy.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 25 mg/kg bw/day, food consumption from days 12 to 15 of pregnancy was reduced in comparison with the control group. However, although there was statistically significant (p < 0.05) variation amongst the groups, the difference between the group treated at 25 mg/kg bw/day and the control group was not statistically significant. Food consumption from days 15 to 18 of pregnancy in this group was slightly lower than the control group, but the difference was not statistically significant.
There was no effect of treatment at 7.5 or 15 mg/kg bw/day on maternal food consumption. There was intergroup variation throughout pregnancy, but the females in these two treated groups consumed a similar or slightly greater amount of food than the control 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:

effects observed, treatment-related

Description (incidence and severity):

There was an effect of treatment at 25 mg/kg bw/day on lung with trachea and kidney weights (Table 3 in 'Any other information on results incl. tables'). For all females (including pregnant, not pregnant and those terminated prematurely or found dead) treated at 25 mg/kg bw/day, although mean dead body weight was significantly (p < 0.01) lower than the control group, the mean absolute and body weight-related lung with trachea weights were significantly (p < 0.01) greater than the control group. Body weight-related kidney weight of this group was also slightly, but not statistically significantly, greater than the control group, although absolute kidney weight was slightly lower. For pregnant females only, killed on day 18, treated at 25 mg/kg bw/day, the same pattern of increased body weight-related lung with trachea and kidney weights were observed but the differences were not as great as for all females.
There was no effect of treatment at 7.5 or 15 mg/kg bw/day on lung with trachea or kidney weights. Although there was an apparent slight dose-related increase in body weight-related lung with trachea weights for all females (including pregnant, not pregnant and those terminated prematurely) treated at 7.5 or 15 mg/kg bw/day, this was not observed for pregnant females only, killed on day 18. The apparent increase was because there were more non-pregnant females in these treated groups and with their lower body weights, their organs were slightly heavier in comparison. Absolute and body weight-related kidney weights in these two groups were similar to those of the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Of the five females in the group treated at 25 mg/kg bw/day, that were killed prematurely or found dead, three had no food present in the stomach, the one female found dead had stomach and intestines distended with gas and all five had dark red lung lobes. All five were pregnant and had live foetuses in utero at the time of maternal death. Dark red lung lobes were observed at the scheduled necropsy on day 18 of pregnancy for four other females in this group. No other treatment-related abnormalities were observed for this group. There were no treatment-related abnormalities observed in any of the females from the groups treated at 7.5 or 15 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean post-implantation loss was similar in all groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no effect of treatment at any of the dose levels investigated on the number of live fetuses. At 25 mg/kg bw/day, the mean numbers of implantations and live foetuses were slightly, but not statistically significantly, lower than the control group (Table 4 in 'Any other information on results incl. tables'). However, values were within historical control ranges and therefore the changes were considered not to be biologically significant. The five females in the 25 mg/kg bw/day treatment group that were killed prematurely or found dead had live foetuses in utero at the time of maternal death.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 24, 21, 22 and 19 females pregnant in the control group and groups treated at 7.5, 15 or 25 mg/kg bw/day, respectively (Table 4 in 'Any other information on results incl. tables').

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: test material cation

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

original value presented in study.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: pure test substance

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

recalculated value, expressed as pure test 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):

There was an effect of treatment at 25 mg/kg bw/day on fetal body weight (Table 5 in 'Any other information on results incl. tables'). Mean fetal weights were significantly lower than in the control group (p < 0.01 male fetuses and all fetuses, p < 0.01- female fetuses) and, consequently, mean gravid uterus weight was significantly (p < 0.01) lower than the control group.
At 7.5 or 15 mg/kg/day, fetal weights and gravid uterus weights were slightly, but not statistically significantly, lower than the control group. However, mean values were within historical control ranges and therefore the changes were considered not to be biologically significant.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the sex ratio of fetuses with near equal proportions of male and female fetuses in each group (Table 4 in 'Any other information on results incl. tables').

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Since fetal body weights in the 25 mg/kg bw/day dose group were significantly lower than in the control group, mean gravid uterus weight was significantly (p < 0.01) lower than the control group (Table 5 in 'Any other information on results incl. tables'). At 7.5 or 15 mg/kg bw/day, fetal weights and therefore gravid uterus weights were slightly, but not statistically significantly, lower than the control group. However, mean values were within historical control ranges and therefore the changes were considered not to be biologically significant.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

MAJOR ABNORMALITIES
No external major abnormalities without skeletal origin were observed.

MINOR ABNORMALITIES
There was no dose-related trend and values were within historical control range, there was considered to be no effect of treatment on the incidence of minor external abnormalities.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

MAJOR ABNORMALITIES
The incidence of fetuses or litters with major abnormalities did not show a treatment-related increase. One fetus in the group treated at 7.5 mg/kg bw/day showed spina bifida and in the group treated at 15 mg/kg bw/day, one showed cleft palate. These abnormalities were considered to be spontaneous and unrelated to treatment.

MINOR ABNORMALITIES
There was no statistically significant or dose-related increase in the numbers of fetuses or litters with minor skeletal abnormalities or in the numbers of fetuses or litters with any particular type of minor skeletal abnormalities. All incidences were within historical control ranges .

VARIATIONS
There was a statistically significant (p < 0.01) increase in the numbers of fetuses in the 25 mg/kg bw/day group with six or less caudal centra, and astragalus not ossified. There was a slight, but not statistically significant, increase in the incidence of fetuses with three or less caudal neural arches. The incidences of these findings in this group were above historical control ranges. There was also a slight, but not statistically significant, increase in the numbers of fetuses in the 25 mg/kg bw/day group with retarded ossification of the occipital and with one or more sternebrae bilobed, bipartite, misaligned or mis-shapen.
The incidences of these findings were with historical control ranges. There were significantly (p < 0.05) greater numbers of litters in the 25 mg/kg bw/day group affected with retarded ossification of the occipital, astragalus not ossified and six or less caudal centra. These findings were considered to be as a result of the lower fetal weights in the 25 mg/kg bw/day group. The incidence of fetuses with the variant of six or less caudal centra ossified was unusually low in the control group and, in comparison, the incidences in the groups treated at 7.5 or 15 mg/kg bw/day appeared high. However the values in these treated groups were within the historical control range and were close to the historical mean so it was concluded that there was no association with treatment.
There was a higher number of fetuses from all treated groups with 14 thoracic vertebrae and five lumbar vertebrae in comparison with the control group. The numbers of fetuses affected were significantly (p < 0.05) different from the control group in the 15 and 25 mg/kg bw/day groups, for both findings but the numbers of litters affected did not show statistically significant or dose-related increases. There were also higher numbers of fetuses with vestigial and/or extra 14th ribs. The numbers of fetuses affected was significantly greater than the control group for unilateral or bilateral vestigial 14th (p < 0.01 7.5 mg/kg bw/day, p < 0.05 25 mg/kg bw/day) and unilateral or bilateral extra 14th rib (p < 0.05 - 15 or 25 mg/kg bw/day). Consequently, there was a lower number of fetuses and litters in the treated groups with 13 normal pairs of ribs and, for numbers of fetuses, the difference from the control group was significant (p < 0.01) for all treated groups.
These five variants are all related. The fetuses show the same numbers of vertebrae but the presence of extra or vestigial ribs classified the vertebra at the thoraco-lumbar border as thoracic instead of lumbar; hence the shift in the proportions. The thoraco-lumbar border is an extremely labile region and so fluctuations in incidences are not unexpected. As a consequence of this, and also since the incidences of these variants were within or only slightly above historical control ranges and no dose-related trends were observed, the findings were considered to be fortuitous and unrelated to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MAJOR ABNORMALITIES
The incidence of fetuses or litters with major abnormalities did not show a treatment-related increase. One fetus in the group treated at 15 mg/kg bw/day showed kidney agenesis. This abnormality was considered to be spontaneous and unrelated to treatment.

MINOR ABNORMALITIES
There was a significantly higher number of fetuses with minor external/visceral abnormalities in the 7.5 and 15 mg/kg bw/day groups (p < 0.01 and p < 0.05, respectively) and a significantly (p < 0.01) higher number of litters affected in the 7.5 mg/kg bw/day group in comparison with the control group. However, the numbers of fetuses and litters affected in the 25 mg/kg bw/day group was not statistically different from the control group. Since there was no dose-related trend and values were within historical control range, there was considered to be no effect of treatment on the incidence of minor visceral abnormalities.
The higher numbers in the 7.5 and 15 mg/kg bw/day groups were due to significantly greater numbers of fetuses with increased renal pelvic cavitation (p < 0.01 and p < 0.05, respectively). There was also a slightly greater number of fetuses with this minor abnormality in the 25 mg/kg bw/day group, in comparison with the control group. There was a similar, less pronounced, trend in the number of litters affected, but statistical significance was not observed. Although the incidence of this abnormality in the treated groups was above the historical control range, there was no dose-related trend so this finding was considered to be fortuitous and unrelated to treatment.

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Remarks:

original value presented in study

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Remarks:

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

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

The findings are in line with retardation of embryonic/foetal growth.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 other: mg/kg bw/day (actual dose received) recalculated value, expressed as pure test substance, see ‘Any other information on results incl. tables’ for respective calculation).

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Verification of dosing solution

The results of analysis of dosing solutions demonstrated that they were prepared correctly and that they were stable for up to one month, which exceeded the period of use, when stored at room temperature in the dark.

 

Calculation of key result maternal and developmental toxicity

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 15 mg test substance cation / kg bw = 20.7 mg pure pure test substance/ kg bw

(100/72.4) x 25 mg test substance cation / kg bw = 34.5 mg pure pure test substance/ kg bw

Table 1. Group maternal body weights (g) – (mean ± SD).

Day of gestation   Group	n	0	6	7	8	9	12	15	18	18#
1	24	27.9 ± 1.7	31.0 ± 1.8	32.2 ± 1.9	33.1 ±1.7	33.9 ± 1.8	39.9 ± 2.1	47.9 ± 3.0	59.0 ± 4.1 (23)	34.7 ± 2.6 (21)
2	21	28.1 ± 1.6	30.9 ± 1.6	31.9 ± 1.6	32.5 ± 1.7	33.3 ± 1.7	39.1 ± 2.3	46.8 ± 3.0	58.3 ± 4.2	34.3 ± 2.6 (19)
3	21	28.0 ± 2.0	31.0 ± 2.2	31.9 ± 2.0	32.6 ± 2.1	33.5 ± 2.2	38.9 ± 3.0	47.0 ± 3.7	58.9 ± 5.6	35.2 ± 3.2 (21)
4	19	28.4 ±1.8	31.7 ± 2.6	32.2 ± 2.1	32.9 ± 1.9	33.9 ± 1.8	39.5 ± 2.5	44.8 ± 3.5 **	53.7 ± 3.9 (14) **	33.9 ± 3.1 (13)
Analysis of variance		NS	NS	NS	NS	NS	NS	p<0.05	p<0.01	NS

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

# = adjusted for gravid uterus weight

Table 2. Group maternal body weights (g) – (mean ± SD).

Day of gestation   Group	n	0 - 6	6 - 9	9 - 12	12 - 15	15 - 18	6 - 15	0 - 18	0 - 18#
1	24	3.1 ± 1.1	2.9 ± 0.6	6.0 ± 0.8	8.0 ± 1.2	11.3 ± 1.7 (23)	16.9 ± 2.1	31.3 ± 3.4 (23)	7.1 ± 2.3 (21)
2	21	2.9 ± 0.9	2.3 ± 0.6	5.9 ± 1.4	7.7 ± 1.3	11.4 ± 1.5	15.9 ± 2.4	30.2 ± 4.1	6.4 ± 2.7 (19)
3	21	3.0 ± 0.8	2.5 ± 0.7	5.4 ± 1.2	8.1 ± 1.0	11.5 ± 2.5	16.1 ± 1.9	30.5 ± 4.2	7.2 ± 2.0 (21)
4	19	3.3 ± 1.7	2.3 ± 1.4	5.6 ± 1.3	5.3 ± 3.2**	8.0 ± 2.5** (14)	13.1 ± 3.9**	25.4 ± 3.9 ** (14)	5.8 ± 2.9 (13) **
Analysis of variance		NS	NS	NS	p<0.01	p<0.01	p<0.01	p<0.01	NS

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

# = adjusted for gravid uterus weight

Table 3. Group mean maternal absolute (g) and body weight related organ weights (g/kg) of pregnant females killed on day 18 – (mean ± SD).

			Absolute weight (g)		Body weight related (g/kg)
Group	n	Dead body weight (g)	Lungs & trachea	Kidney total	Lungs & trachea	Kidney total
1	23	57.6 ± 4.2	0.244 ± 0.032	0.528± 0.042	4.276 ± 0.591	9.191 ± 0.789
2	21	57.1 ± 4.2	0.249 ± 0.023	0.529 ± 0.043	4.508 ± 0.753	9.302 ± 0.767
3	21	57.8 ± 5.3	0.250 ± 0.034	0.527 ± 0.043	4.338 ± 0.598	9.163 ± 0.837
4	14	52.7 ± 4.1 **	0.271 ± 0.038	0.515 ± 0.075	5.155 ± 0.770 **	9.763 ± 1.102
Analysis of variance		p<0.01	NS	NS	p<0.01	NS

n = number of animals in mean data

** = significantly different from control, p<0.01 Student’s t-test

 

Table 4. Group mean pregnancy data

Group	Number of pregnant/mated	Mean no. of implantations ± SD	Mean no. of live fetuses ± SD	Mean post-implantation loss (%)	Sex ratio (%) M : F
1	24/26	13.7 ± 2.0	13.0 ± 2.3	5.5	55:45
2	21/26	13.5 ± 2.1	12.7 ± 2.5	6.2	48:52
3	22/26	13.9 ± 2.1	13.0 ± 2.3	6.3	48:52
4	19/26	12.3 ± 2.9	11.7 ± 3.0	5.0	51:49
Analysis of variance		NS	NS
Kruskal-Wallis test				NS	NS

 

 

Table 5. Group mean fetal gravid uterus weights (g) – (mean ± SD)

Group	n	Fetuses (male and female)	Uterus
1	23	1.41 ± 0.08	24.1 ± (21)
2	21	1.38 ± 0.09	23.8 ± 3.3 (19)
3	22	1.37 ± 0.08	23.7 ± 3.7 (21)
4	14	1.28 ± 0.16**	20.0 ± 2.6(13) **
Analysis of variance		p<0.01	p<0.01

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

 

Conclusions:

In this GLP compliant developmental toxicity study, performed similar to an OECD 414 test protocol, maternal NOAEL of 15 mg test substance cation/kg bw/day can be set based on clinical signs, body weight and gross pathology. The developmental NOAELs of 15 mg test substance cation/kg bw/day can be set based decreases in mean fetal weights and retarded ossification of the occipital, increases in the number with 6 caudal centra, increases in the number with uni- or bilateral extra 14th ribs and increases in the number with non-ossified astragalus in the hindlimb seen at 25 mg test substance cation/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity are 20.7 mg pure test substance/kg bw/day. The developmental effects are considered secondary to the maternal effects.

Executive summary:

In this GLP compliant developmental toxicity study, performed similar to a study according to OECD TG 414, the test substance was tested for embryonic and fetotoxic effects in Crl:CD-1 mice. The test material was administered by gavage (10 mL/kg bw) in an aqueous solution of purified water at daily doses of 7.5, 15 and 25 mg test substance cation/kg body weight from gestation day 6 to 15. The control group was dosed with purified water only. Each group consisted of 26 time-mated mice (Crl:CD-1 (ICR)BR). Maternal clinical signs, body weights and food consumption were recorded. On day 18 of gestation the females were killed. All females were subject to necropsy at which lungs with trachea and the kidneys were removed and weighed. Pregnancy status was assessed and the gravid uterus weight and numbers of live and dead implantations were recorded. Live fetuses were weighed and, after light fixation in alcohol (70%), were examined for external abnormalities and sexed. One half of the fetuses were replaced in 70% alcohol and then examined for visceral abnormalities and eviscerated before being cleared in potassium hydroxide, stained with alizarin red S and examined for skeletal abnormalities. The remaining fetuses were fixed in Bouin's fluid and were examined for visceral abnormalities.

In the 25 mg/kg bw/day group, there was an effect of treatment on maternal clinical condition. Four females were affected and were consequently terminated prematurely between days 15 and 17 of pregnancy. The effects included piloerection, laboured respiration, hunched posture, hypothermia, hypoactivity and pale extremities and eyes. One female in this group was found dead on day 16 of pregnancy, but no changes in clinical condition were observed prior to death. There was an effect of treatment on maternal body weight and food consumption. Body weight gain was retarded and food consumption was reduced, in comparison with the control group, from approximately day 12 of pregnancy until necropsy on day 18. The four females terminated prematurely, the one female found dead and four other females in this group had dark red lung lobes at necropsy. Three females also had no food present in the stomach. Lung and kidney weights were affected by treatment. Absolute and body weight-related lung with trachea weights and body weight-related kidney weights were greater than in the control group.

The numbers of implantations or live fetuses, post-implantation loss or fetal sex ratio were not affected by treatment. Retardation of embryonic/fetal growth was seen in the 25 mg/kg bw/day group. Mean fetal weight was lower than in the control group, and there was an increase in the incidence of certain variants of skeletal ossification (retarded ossification of the caudal vertebrae, occipital and astragalus, and bilobed, bipartite, misaligned and mis-shapen sternebrae). There was no evidence of teratogenicity at this dose level.

In the 7.5 and 15 mg/kg bw/day groups, there was no effect of treatment on clinical condition, body weight or food consumption of dams and there were no premature deaths. There were no treatment-related abnormalities at necropsy. Lung and kidney weights were not affected by treatment. There was no effect of treatment at this dose level on the numbers of implantations or live fetuses, post-implantation loss of fetal sex ratio. Retardation of embryonic/fetal growth was not seen in this group and there was no evidence of teratogenicity.

Maternal toxicity and developmental toxicity were seen at the high dose group (25 mg test substance cation/kg bw bw/day). The maternal NOAEL of 15 mg test substance cation/kg bw/day can be set based on clinical signs, reduced body weight and gross pathology. The developmental NOAELs of 15 mg test substance cation/kg bw/day can be set based on decreases in mean fetal weights and retarded ossification of the occipital, increases in the number with 6 caudal centra, increases in the number with uni- or bilateral extra 14th ribs and increases in the number with non-ossified astragalus in the hindlimb seen at  a dose of 25 mg test substance cation/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity is mg pure test substance/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity is 20.7 mg pure test substance/kg bw/day. The developmental effects are considered secondary to the maternal toxicity seen in the high dose group.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 1992 to June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar-derived (Alpk:APfSD) (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: The majority of rats were within the weight range 220 to 270 g (extreme range 209 to 297 g).
- Fasting period before study: No
- Housing: Each rat was individually housed. The cages had one solid stainless steel side, the other side, floor, back and front were constructed of stainless steel mesh. The internal measurements were 26.5 x 50.0 x 20.7 cm with a floor area of 1325 cm2. The cages were suspended over collecting trays cleaned by an automatic flushing system.
- Diet: ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70 (occasional excursions 39 to 82)
- Air changes (per hr): 25 to 30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
May 1992 to June 1992

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionised

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were formulated in deionised water and the concentration was adjusted to give a constant volume of 1 mL/100 g body weight for each dose level. An appropriate amount of deionised water was added to a weighed amount of test substance to provide one dosing solution per dose level. Each dosing solution was then thoroughly mixed before being divided into aliquots. The control article was also dispensed into aliquots. The aliquots were stored in the dark at room temperature and fresh bottles were used for each day of the study.

VEHICLE
- Amount of vehicle (if gavage): 1 mL/100 g body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each dosing solution was analysed prior to the start of dosing to verify the achieved concentrations of the test substance. The chemical stability of the test substance in deionised water was determined by re-analysis of the low and high dose formulations after an interval of 32 days. The diluted solutions were analysed by high performance liquid chromatography with UV detection at 255 nm. The sample concentrations were calculated by reference to a single calibration standard.
The homogeneity of the test substance in water was assumed as all formulations were solutions.

Details on mating procedure:

- Impregnation procedure: cohoused, with unrelated male from the same strain.
- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight.
- Proof of pregnancy: Sperm in vaginal smear referred to as day 1 of gestation.

Duration of treatment / exposure:

Days 7-16 (inclusive) of gestation

Frequency of treatment:

Daily

Duration of test:

Day 22 of gestation

Dose / conc.:

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

Remarks:

Group 2, low dose group. Concentration of test substance cation.

Dose / conc.:

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

Remarks:

Group 3, mid dose group. Concentration of test substance cation.

Dose / conc.:

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

Remarks:

Group 4, high dose group. Concentration of test substance cation.

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected from the results of a previous developmental toxicity study in rat. In this study, there was substantial maternal mortality at 10 mg test substance/kg/day and therefore 8 mg test substance/kg/day was selected as the maximum tolerated dose ie. the highest dose which would not affect mortality.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily. Any changes in behaviour or clinical condition were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule: 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: Yes
- Sacrifice on gestation day 22
- Organs examined: Tissues were examined macroscopically. The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
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. In addition, each fetus was weighed and individually identified within the litter by means of a cardboard tag.

Fetal examinations:

- External examinations: Yes: all per litter. Each fetus was examined for external abnormalities and for cleft palate.
- Soft tissue examinations: Yes: all per litter. All foetuses were then examined internally for visceral abnormalities under magnification, sexed, eviscerated and fixed in methanol.
- Skeletal examinations: Yes: all per litter. After examination of the head, the carcasses were stained. 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 six point scale.
- Head examinations: Yes: all per litter. The head of each fetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities.

Statistics:

Data relating to the animals which were non-pregnant were excluded from the statistical analysis.
Maternal body weight during the dosing and post dosing periods was considered by analysis of covariance on initial (day 7) body weight.
Maternal food consumption during the dosing and post dosing periods, the numbers of implantations and live fetuses per female, gravid uterus weight, mean fetal weights per litter and mean manus and pes scores were considered by analysis of variance.
Maternal performance data, the proportion of fetuses with each individual manus and pes score, the proportion of fetuses with each defect and the proportion of litters with each defect were considered by Fisher’s Exact Test.
Pre-implantation loss, post-implantation loss, early intra-uterine deaths, late intra-uterine deaths, major external/visceral defects, minor external/visceral defects, external/visceral variants, major skeletal defects, minor skeletal defects and skeletal variants were analysed as follows: Percentages were analysed by analysis of variance following the double arcsine transformation of Freeman and Tukey. Then, the proportion of fetuses and the proportion of litters affected were considered by Fisher's Exact Test. All statistical tests were two-sided with the exception of the analyses of each individual defect which were one-sided.

Indices:

Percentages of pre-implantation loss and post-implantation loss are calculated (see 'Any other information on materials and methods incl. tables').

Historical control data:

Historical control data – fetus based incidence of findings.
Brain – internal hydrocephaly: Mean 0.1, SD 0.3, N 12, range 0.0 – 0.9
Ureter – slightly dilated: Mean 1.2, SD 1.6, N=12, range 0.0 – 5.5
Extra (14th) ribs – short: Mean 8.1, SD 4.3, N=12, Range 2.3 – 14.0

Historical control data – litter based incidence of findings.
Brain – internal hydrocephaly: Mean 1.5, SD 3.5, N 12, range 0.0 – 9.1

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of clinical findings was low and was considered to be unrelated to the test compound.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortalities during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a mean body weight loss at high dose group between the first and second days of dosing (Days 7-8) while other groups including control showed a mean weight gain (Table 1 in ‘Any other information on results incl. tables’). There was considerable individual variation of the size of the effect with a maximum weight loss of 10 g for one female but with some animals unaffected. Nevertheless, this is considered to be an effect of compound because of the increased numbers of pregnant animals, in total 9, in the high dose group lost weight compared with the control group where only 3 animals were affected. In addition, where weight loss did occur, it was generally greater than in the controls: 8 top dose animals had a weight loss of >=4 g compared with a weight loss of 1 or 2 g in the 3 controls. Subsequent gains were generally similar to those of control resulting in marginal reductions for the full dosing period (although giving some statistically significant differences) and for the overall gestation period. There were no compound-related effects on body weight at the other dose levels.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was a marginal difference in food consumption (which did not achieve statistical significance) for the high dose group compared with the control group during Days 7-10. (The group mean body weight was higher than that of other groups at the start of dosing and therefore the animals might have been expected to eat more.) There was no compound-related effect at the other dose levels.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Findings were of a type commonly seen in young female Alpk:APfSD rats and were considered to be unrelated to compound.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Higher pre-implantation loss was observed in the high dose group compared to the control group (Table 2 in ‘Any other information on results incl. tables’). However, as pre-implantation loss occurs before the start of dosing, this is not an effect of compound.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

original value (cation) presented in study.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

recalculated value, expressed as pure test 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

Description (incidence and severity):

There was no clear evidence for an effect on fetal weight although two litters in the high dose group had only one surviving fetus, both being unusually small for a single fetus.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

All pregnant females produced viable fetuses at termination. The number of live offspring was slightly lower in the high dose group because of higher pre-implantation loss in this dose group compared to the control group (Table 2 in ‘Any other information on results incl. tables’). However, as pre-implantation loss occurs before the start of dosing, this is not an effect of the compound.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio was similar in the control and treatment groups (Table 2 in ‘Any other information on results incl. tables’).

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Litter weights were slightly lower for the high dose group because of a higher pre-implantation loss in this dose group compared with the control group (Table 2 in ‘Any other information on results incl. tables’). However, as pre-implantation loss occurs before the start of dosing, this is not an effect of the compound.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external malformations were observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The overall incidence of minor skeletal defects was higher on a fetal basis in the low and high dose groups. This was largely due to 5th sternebra bipartite. Additionally, there were increases in centra which were not ossified for a number of different cervical vertebrae in the low dose group. A higher incidence of partially ossified parietals was seen in the low and mid dose groups. In the absence of any dose response or a litter effect, none of these differences is considered to be compound-related.
There were no effects on the overall incidence of skeletal variants. The incidence of extra (14th) ribs was higher than control in all groups on a fetal basis but was comparable to that of historical controls, with no effect on a litter basis. Slightly higher incidences of not ossified odontoid and not ossified 5th sternebra were seen in the low dose group but in the absence of a dose response or an effect on a litter basis, this is considered not to be an effect of compound.
An overview of the incidence of skeletal malformation and variations can be found in Table 3 in ‘Any other information on results incl. tables’.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of fetuses with major defects was 1, 4, 1 and 0 in the control, low, mid and high dose group respectively. Three fetuses from the low dose group (two from the same litter) had internal hydrocephaly. There was no association with dose and neither the type nor incidence of major abnormalities was considered to be associated with administration of the test substance. This defect has been observed at a similar frequency in the historical data of this Laboratory with two affected control litters.
There was no evidence of any compound-related effect on the overall incidence of minor visceral defects. The only individual defect to show a statistically significant increase was slightly dilated ureters at the high dose group. However, the overall incidence was low and within the historical control range and is therefore considered to be unrelated to compound.
The only visceral variant recorded on this study was kinked ureter but the incidence was not compound- or dose-related.
An overview of the incidence of visceral malformation and variations can be found in Table 3 in ‘Any other information on results incl. tables’.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Slightly higher manus scores were observed in the low and high dose group (Table 4 in ‘Any other information on results incl. tables’). The overall mean litter scores, which are a good indicator of retarded fetal development, did not achieve statistical significance compared with control.

Dose descriptor:

NOAEL

Effect level:

>= 8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

original value presented in study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 11 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Verification of dosing solution

The concentrations of the test substance in deionised water were within 8% of nominal. The stability of the test substance in aqueous solutions was shown to be satisfactory over a 32 day period which exceeded the period of use.

Calculation of key result maternal and developmental toxicity

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 3 mg test substance cation / kg bw = 4.1 mg pure pure test substance/ kg bw.

(100/72.4) x 8 mg test substance cation / kg bw = 11.0 mg pure pure test substance/ kg bw.

Table 1. Maternal body weight.

Gestation day		Dose level (mg/kg bw/day)
Pre-dosing		Control	1	3	8
1	Mean	250.7	248.0	241.7	252.4
	SD	18.2	21.0	13.5	16.4
	N	24	21	24	22
4	Mean	268.7	267.1	260.5	272.2
	SD	19.3	21.0	16.2	16.8
	N	24	21	24	22
7	Mean	282.3	278.9	272.4	287.0
	SD	18.3	21.1	16.8	17.9
	N	24	21	24	22
During dosing
8	Mean	285.3	281.7	275.0	286.3
	SD	18.2	21.4	17.9	17.2
	N	24	21	24	22
	Adjusted mean	283.1	282.8	282.5	279.2**
9	Mean	289.5	285.5	278.1	288.7
	SD	19.4	21.9	18.0	17.7
	N	24	21	24	22
	Adjusted mean	287.3	286.5	285.8	281.7**
10	Mean	292.9	289.3	283.8	294.8
	SD	19.4	22.2	18.4	18.5
	N	24	21	24	22
	Adjusted mean	290.6	290.0	291.4	287.3**
11	Mean	297.7	293.7	289.7	300.4
	SD	19.2	23.5	18.5	19.5
	N	24	21	24	22
	Adjusted mean	295.4	294.4	297.7	292.6*
12	Mean	303.3	299.5	294.8	304.5
	SD	18.2	23.7	18.7	18.3
	N	24	21	24	22
	Adjusted mean	301.0	300.3	302.5	297.1**
13	Mean	308.3	304.4	298.9	310.4
	SD	18.5	24.6	19.7	18.9
	N	24	21	24	22
	Adjusted mean	305.9	305.2	307.1	302.3*
14	Mean	314.3	309.1	304.8	316.0
	SD	18.7	24.2	19.2	19.4
	N	24	21	24	22
	Adjusted mean	311.9	310.4	312.9	307.9*
15	Mean	319.3	315.7	311.3	321.3
	SD	19.7	23.7	19.0	18.5
	N	24	21	24	22
	Adjusted mean	317.0	316.6	319.3	313.5
16	Mean	325.0	320.1	316.5	325.8
	SD	19.4	24.1	18.7	18.8
	N	24	21	24	22
	Adjusted mean	322.6	321.2	324.4	318.1*
Post-dosing
19	Mean	353.5	349.9	348.4	355.2
	SD	21.6	26.2	19.6	23.7
	N	24	21	24	22
	Adjusted mean	351.1	351.6	356.6	347.2
22	Mean	390.8	385.1	386.5	387.9
	SD	30.5	33.0	20.5	29.1
	N	24	21	24	22
	Adjusted mean	388.1	387.4	395.7	379.2

* Statistically significant difference from control at the 5% level.

** Statistically significant difference from control at the 1% level

Table 2. Litter data.

			dose level (mg/kg bw/day)
			Control		1		3		8
Pregnant/mated		24/24		21/24		24/24		22/24
Live fetuses in utero at termination		24/24		21/24		24/24		22/24
Mean no. of corpora lutea	Mean	13.2		13.2		13.3		13.2
	SD	2.6		2.2		1.5		2.5
	N	24		21		24		22
Mean no. of implantations	Mean	11.1		10.2		11.7		10.0
	SD	3.9		3.7		2.6		4.2
	N	24		21		24		22
Mean no. of live fetuses	Mean	10.7		9.8		11.4		9.4
	SD	3.9		3.6		2.7		4.5
	N	24		21		24		22
Mean gravid uterus weight (g)	Mean	72.6		65.7		75.7		64.2
	SD	25.0		22.8		17.7		28.8
	N	24		21		24		22
Mean litter weight (g)	Mean	50.1		45.3		52.4		44.0
	SD	18.6		15.9		12.4		21.6
	N	24		21		24		22
Mean fetal weight (g)	Mean	4.77		4.75		4.62		4.61
	SD	0.59		0.66		0.40		0.55
	N	24		21		24		22
Sex distribution
Proportion of male fetuses		125/256		99/206		135/273		96/206
Percentage	Mean	48.4		46.7		48.7		45.1
	SD	19.3		16.5		14.6		21.6
	N	24		21		24		22
Pre-implantation loss
Proportion of implants affected		51/317		63/277		39/320		69/290*
Percentage	Mean	17.2		23.7		12.4		26.0
	SD	25.4		24.2		15.4		26.1
	N	24		21		24		22
Proportion of litter affected		16/24		18/21		16/24		17/22
Post-implantation loss
Proportion of implants affected		10/266		8/214		8/281		15/221
Percentage	Mean	5.0		3.1		2.9		8.3
	SD	11.6		6.8		6.0		19.7
	N	24		21		24		22
Proportion of litter affected		6/24		5/21		5/24		5/22
Early intra-uterine deaths
Proportion of implants affected		10/266		8/214		7/281		15/221
Percentage	Mean	5.0		3.1		2.5		8.3
	SD	11.6		6.8		5.2		19.7
	N	24		21		24		22
Proportion of litter affected		6/24		5/21		5/24		5/22
Late intra-uterine deaths
Proportion of implants affected		0/266		0/214		1/281		0/221
Percentage	Mean	0,0		0.0		0.4		0.0
	SD	0,0		0,0		1.9		0.0
	N	24		21		24		22
Proportion of litter affected		0/24		0/21		1/24		0/22

* Statistically significant difference from control at the 5% level.

** Statistically significant difference from control at the 1% level.

 

Table 3. Fetal defects and variants

Major external/visceral defects
Proportion of fetuses affected		1/256	4/206	1/273	0/206
Percentage	Mean	0.4	1.5	0.4	0.0
	SD	4.0	1.9	1,9	0,0
	N	24	21	24	22
Proportion of litter affected		1/24	3/21	1/24	0/22
Minor external/visceral defects only
Proportion of fetuses affected		2/256	6/206	1/273	6/206
Percentage	Mean	4.6	3.5	0.3	6.4
	SD	20,4	6.6	1.5	21.7
	N	24	21	24	22
Proportion of litter affected		2/24	6/21	1/24	4/22
External/visceral variants
Proportion of fetuses affected		16/256	19/206	14/273	12/206
Percentage	Mean	13.3	14.0	4.9	14.5
	SD	18,2	23.3	6.8	29.6
	N	24	21	24	22
Proportion of litter affected		9/24	10/21	10/24	8/22
Major skeletal defects
Proportion of fetuses affected		0/256	0/206	0/273	0/206
Percentage	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	24	21	24	22
Proportion of litter affected		0/24	0/21	0/24	0/22
Minor skeletal defects only
Proportion of fetuses affected		71/256	86/206**	80/273	67/206
Percentage	Mean	26.9	39.1	29.6	37.5
	SD	23.2	24.6	17.1	25.7
	N	24	21	24	22
Proportion of litter affected		21/24	18/21	23/24	21/22
Skeletal variations
Proportion of fetuses affected		215/256	184/206	238/273	180/206
Percentage	Mean	85.6	86.2	87.1	87.9
	SD	20.0	26.8	16.5	17.3
	N	24	21	24	22
Proportion of litter affected		24/24	20/21	24/24	22/22

* Statistically significant difference from control at the 5% level.

** Statistically significant difference from control at the 1% level.

 

Table 4. Manus/Pes assessment scores.

		Dose level (mg/kg bw/day)
Manus scores		Control	1	3	8
Proportion with score 3		4/256	1/206	2/273	1/206
Proportion with score 4		168/256	104/206**	166/273	116/206
Proportion with score 5		77/256	99/206**	104/273	88/206**
Proportion with score 6		7/256	2/206	1/273	1/206
Mean manus score per litter	Mean	4.36	4.45	4.38	4.54
	SD	0.45	0.41	0.37	0.49
	N	24	21	24	22
Pes score
Proportion with score 3		1/256	0/206	0/273	0/206
Proportion with score 4		16/256	9/206	14/273	10/206
Proportion with score 5		229/256	179/206	245/273	190/206
Proportion with score 6		10/256	18/206*	14/273	6/206
Mean pes score per litter	Mean	4.98	4.99	4.99	5.03
	SD	0.25	0.33	0.20	0.24
	N	24	21	24	22

* Statistically significant difference from control at the 5% level.

** Statistically significant difference from control at the 1% level.

 

Conclusions:

In this GLP compliant developmental toxicity study, performed similar to OECD 414, maternal NOAEL is found to be 3 mg test substance cation/kg bw/day (recalculated NOAEL is 4.1 mg pure test substance/kg bw/day). The NOAEL for developmental effects is greater than the highest dose tested (8 mg test substance cation/kg bw/day), because no treatment-related adverse developmental effects were seen at this dose. Therefore, the NOAEL for developmental toxicity is considered to be higher than 8 mg test substance cation/kg bw/day (recalculated NOAEL is 11.0 mg pure test substance/kg bw/day).

Executive summary:

In this GLP compliant developmental toxicity study, performed similar to a study in accordance with OECD TG 414, the test substance was tested for embryonic and fetotoxic effects in Wistar-derived rats. The test material was administered by gavage (1 mL/100 g bw) in an aqueous solution of deionised water at daily doses of 1, 3 and 8 mg/kg body weight from gestation day 7 to 16. The control group was dosed with deionised water only. Each group consisted of 24 pregnant Wistar-derived rats. On day 22 of gestation the females were killed and their uteri examined for live fetuses and intra-uterine deaths. The fetuses were weighed, examined for external/visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

There was evidence of a compound-related effect on maternal bodyweight at the high dose group (8 mg test substance cation/kg bw/day) and it is considered that this closely approaches the maximum tolerated dose in the pregnant rat. There were no treatment related macroscopic findings in the pregnant females. At the high dose group, there was a reduction in fetal weight and a reduction in the number of live fetuses due to increased pre-implantation losses and early deaths. Pre-implantation loss is not considered to be a treatment-related effect, since pre-implantation loss occurs before dosing. There was no increase in the overall numbers of fetuses with defects, anomalies or major skeletal effects. Hydrocephaly was seen in 3 fetuses from 2 litters from the low dose group (1mg/kg bw/day), an incidence outside the historical control range but not repeated at higher doses. Dilated ureter was increased in the high dose group (2.9% with 0.4% in control). The incidence of short 14th ribs was increased, without a dose-response, in all treated groups compared to concurrent controls, but were within the historical control values. There were no other significant dose related findings.

Maternal toxicity was seen at the high dose group (8 mg test substance cation/kg bw/day). The maternal NOAEL is found to be 3 mg test substance cation/kg bw/day (recalculated NOAEL is 4.1 mg pure test substance/kg bw/day). The NOAEL for developmental effects was greater than the highest dose applied, as no treatment-related adverse developmental effects were seen at 8 mg test substance cation/kg bw/day, the highest dose tested. Therefore, the NOAEL for developmental toxicity is considered to be higher than 8 mg test substance cation/kg bw/day (recalculated NOAEL is 11.0 mg pure test substance/kg bw/day).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

11 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP compliant, similar to OECD 414.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental toxicity - Rat

In this GLP compliant developmental toxicity study, performed similar to an OECD TG 414 protocol, the test substance was tested for embryonic and fetotoxic effects in rats. The test material was administered by gavage (1 mL/100 g bw) in an aqueous solution of deionised water at daily doses of 1, 3 and 8 mg/kg body weight from gestation day 7 to 16. The control group was dosed with deionised water only. Each group consisted of 24 pregnant Wistar-derived rats. On day 22 of gestation the females were killed and their uteri examined for live fetuses and intra-uterine deaths. The fetuses were weighed, examined for external/visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

There was evidence of a compound-related effect on maternal bodyweight at the high dose group (8 mg test substance cation/kg bw/day) and it is considered that this closely approaches the maximum tolerated dose in the pregnant rat. There were no treatment related macroscopic findings in the pregnant females. At the high dose group, there was a reduction in fetal weight and a reduction in the number of live fetuses due to increased pre-implantation losses and early deaths. Pre-implantation loss is not considered to be a treatment-related effect, since pre-implantation loss occurs before dosing. There was no increase in the overall numbers of fetuses with defects, anomalies or major skeletal effects. Hydrocephaly was seen in 3 fetuses from 2 litters from the low dose group (1 mg/kg bw/day), an incidence outside the historical control range but not repeated at higher doses. Dilated ureter was increased in the high dose group (2.9% with 0.4% in control). The incidence of short 14th ribs was increased, without a dose-response, in all treated groups compared to concurrent controls, but were within the historical control values. There were no other significant dose related findings.

Maternal toxicity was seen at the high dose group (8 mg test substance cation/kg bw/day). The maternal NOAEL is found to be 3 mg test substance cation/kg bw/day (recalculated NOAEL is 4.1 mg pure test substance/kg bw/day). The NOAEL for developmental effects was treater than the highest dose applied, because no treatment-related adverse developmental effects were seen at 8 mg test substance cation/kg bw/day, the highest dose tested. Therefore, the NOAEL for developmental toxicity is considered to be higher than 8 mg test substance cation/kg bw/day (recalculated NOAEL is 11.0 mg pure test substance/kg bw/day).

Developmental toxicity - Mice

In this GLP compliant developmental toxicity study, performed similar to the OECD TG 414 protocol, the test substance was tested for embryonic and fetotoxic effects in mice. The test material was administered by gavage (10 mL/kg bw) in an aqueous solution of purified water at daily doses of 7.5, 15 and 25 mg test substance cation/kg body weight from gestation day 6 to 15. The control group was dosed with purified water only. Each group consisted of 26 time-mated mice (Crl:CD-1 (ICR)BR). Maternal clinical signs, body weights and food consumption were recorded. On day 18 of gestation the females were killed. All females were subject to necropsy at which lungs with trachea and the kidneys were removed and weighed. Pregnancy status was assessed and the gravid uterus weight and numbers of live and dead implantations were recorded. Live fetuses were weighed and, after light fixation in alcohol (70%), were examined for external abnormalities and sexed. One half of the fetuses were replaced in 70% alcohol and then examined for visceral abnormalities and eviscerated before being cleared in potassium hydroxide, stained with alizarin red S and examined for skeletal abnormalities. The remaining fetuses were fixed in Bouin's fluid and were examined for visceral abnormalities.

In the 25 mg/kg bw/day group, there was an effect of treatment on maternal clinical condition. Four females were affected and were consequently terminated prematurely between days 15 and 17 of pregnancy. The effects included piloerection, laboured respiration, hunched posture, hypothermia, hypoactivity and pale extremities and eyes. One female in this group was found dead on day 16 of pregnancy, but no changes in clinical condition were observed prior to death. There was an effect of treatment on maternal body weight and food consumption. Body weight gain was retarded and food consumption was reduced, in comparison with the control group, from approximately day 12 of pregnancy until necropsy on day 18. The four females terminated prematurely, the one female found dead and four other females in this group had dark red lung lobes at necropsy. Three females also had no food present in the stomach. Lung and kidney weights were affected by treatment. Absolute and body weight-related lung with trachea weights and body weight-related kidney weights were greater than in the control group.

The numbers of implantations or live fetuses, post-implantation loss or fetal sex ratio were not affected by treatment. Retardation of embryonic/fetal growth was seen in the 25 mg/kg bw/day group. Mean fetal weight was lower than in the control group, and there was an increase in the incidence of certain variants of skeletal ossification (retarded ossification of the caudal vertebrae, occipital and astragalus, and bilobed, bipartite, misaligned and mis-shapen sternebrae). There was no evidence of teratogenicity at this dose level.

In the 7.5 and 15 mg/kg bw/day groups, there was no effect of treatment on clinical condition, body weight or food consumption of dams and there were no premature deaths. There were no treatment-related abnormalities at necropsy. Lung and kidney weights were not affected by treatment. There was no effect of treatment at this dose level on the numbers of implantations or live fetuses, post-implantation loss of fetal sex ratio. Retardation of embryonic/fetal growth was not seen in this group and there was no evidence of teratogenicity.

Maternal toxicity and developmental toxicity were seen at the high dose group (25 mg test substance cation/kg bw bw/day). The maternal NOAEL of 15 mg test substance cation/kg bw/day can be set based on clinical signs, reduced body weight and gross pathology. The developmental NOAELs of 15 mg test substance cation/kg bw/day can be set based on decreases in mean fetal weights and retarded ossification of the occipital, increases in the number with 6 caudal centra, increases in the number with uni- or bilateral extra 14th ribs and increases in the number with non-ossified astragalus in the hindlimb seen at a dose of 25 mg test substance cation/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity are 20.7 mg pure test substance/kg bw/day. The developmental effects are considered secondary to the maternal toxicity seen in the high dose group.

Justification for classification or non-classification

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