Paraquat-dichloride

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Paraquat-dichloride

EC Number:

217-615-7

EC Name:

Paraquat-dichloride

Cas Number:

1910-42-5

Molecular formula:

C12H14N2.2Cl

IUPAC Name:

1,1’-dimethyl-4,4’-bipyridyldiylium dichloride

Test material form:

liquid

Test animals

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.)

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 ppm

Remarks:

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

Doses / concentrations 2

Dose / conc.:

75 ppm

Remarks:

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

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

1

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'.

2

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

3

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'.

4

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

Target system / organ toxicity (P0)

1

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

2

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Details on results (P1)

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.

Effect levels (P1)

1

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'.

2

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

3

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'.

4

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

Target system / organ toxicity (P1)

1

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

2

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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'.

2

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

3

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'.

4

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

1

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'.

2

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

3

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'.

4

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

5

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'.

6

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

7

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'.

8

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

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.